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Biomedical subjects

P F Watson

Publications and source records attributed to P F Watson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Association of Graves' disease with an allele of the interleukin-1 receptor antagonist gene.

The proinflammatory cytokine, interleukin-1 (IL-1), has been implicated in the pathogenesis of several autoimmune and inflammatory diseases. One of its natural inhibitors, IL-1 receptor antagonist, is a potent antiinflammatory agent. We have previously described genetic associations between an allele of the IL-1 receptor antagonist gene (IL1RN*2) and several autoimmune and inflammatory diseases. In the present study, we tested the association of this polymorphism with thyroid diseases. We genotyped 2 separate cohorts (total of 100 patients) with Graves' disease and 58 patients with Hashimoto's thyroiditis and compared IL1RN*2 frequencies with those in 261 ethnically matched controls. There was a significant increase in IL1RN*2 frequency and carriage rate in Graves' disease, but this was not associated with thyroid antibody levels, T4 levels, thyroid-associated ophthalmopathy, or outcome after antithyroid drug treatment. In contrast, there was no difference in the frequency of IL1RN*2 between patients with Hashimoto's thyroiditis and the control group. Whether the IL1RN polymorphism makes a direct functional contribution to the pathogenesis of Graves' disease or is acting as a marker for a linked gene is being investigated.

Alleles↗
PubMed1995

Lack of association between a polymorphism in the coding region of the thyrotropin receptor gene and Graves' disease.

Using a combination of polymerase chain reaction amplification, oligonucleotide mismatch hybridization, and direct sequencing, we analyzed the distribution of a recently described TSH receptor gene polymorphism in 88 patients with Graves' disease but no clinically apparent eye disease, 53 patients with Graves' disease and associated ophthalmopathy, 39 patients with autoimmune hypothyroidism, and 156 control subjects. No significant difference in the distribution of this polymorphism was found between either group of Graves' patients, the hypothyroid patients, or the control group. These results suggest that this coding region polymorphism is not associated with the occurrence of Graves' disease or Graves' ophthalmopathy.

Alleles↗
PubMed1995

The presence of water channel proteins in ram and human sperm membranes.

Ram and human spermatozoa have a high coefficient of osmotic water permeability (Pf) with a low activation energy (Ea), suggesting the presence of water channels within their plasma membranes. Sperm membranes were examined for the presence of two known water channel proteins, CHIP28 and glucose transporters belonging to the GLUT family of proteins. The water permeability of ram spermatozoa was not inhibited by mercuric chloride to which the CHIP28 channel is sensitive. The CHIP28 protein was not located in western blots of ram sperm membrane preparations that used an anti-CHIP28 antibody. The water permeability of ram and human spermatozoa was inhibited in the presence of phloretin, an inhibitor of glucose transport. Rabbit spermatozoa, which have a low Pf and a high Ea value, suggesting a non-porous membrane, were unaffected by phloretin. These results indicate that the erythrocyte and proximal tubule water channel, CHIP28, is not present in sperm membranes but that sperm membrane glucose transporters may have a secondary water channel function.

Animals↗
PubMed1995

Semi-quantitative analysis of interleukin-1 alpha, interleukin-6 and interleukin-8 mRNA expression by human thyrocytes.

It has been suggested that the thyroid itself may contribute to the inflammatory process observed in autoimmune thyroiditis by releasing the cytokines interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), but studies of cytokine gene expression in thyrocytes have been limited and conflicting. A semi-quantitative reverse transcription-PCR technique has been used to investigate the expression of IL-1 alpha, IL-6 and IL-8 mRNA in the human thyroid cell line HTori3 and in cultures of primary human thyroid follicular cells (TFCs). Cytokine mRNA levels were examined over a 24-h period, and the modulatory effects of exogenous IL-1 alpha, interferon-gamma (IFN-gamma) and TSH investigated. Basal expression of IL-1 alpha, IL-6 and IL-8 mRNA was detected in HTori3 and primary TFC cultures. Stimulation with IL-1 (10 U/ml) for 12 h produced an increase in the level of IL-1 alpha mRNA in both primary TFC and HTori3 cultures. IL-6 and IL-8 mRNA levels were increased by the addition of IL-1 in both cell types, and this effect was detected throughout the 24-h time-course. IFN-gamma (100 U/ml) had no significant effect on cytokine gene expression. A higher concentration of IFN-gamma (500 U/ml) had no significant effect on the expression of IL-1 alpha or IL-8 but produced an increase in the level of IL-6 mRNA in primary cultures and in HTori3 cells. Addition of TSH (1 mU/ml) produced an increase in the level of IL-1 alpha mRNA in primary TFC and HTori3 cells, at 12 and 24 h. TSH had no significant effect on the expression of IL-6 or IL-8 mRNA. These results demonstrate that human TFCs constitutively express IL-1 alpha, IL-6 and IL-8 mRNA and that this expression can be modulated by IL-1, IFN-gamma and TSH.

Base Sequence↗
PubMed1994

Stimulating effect of pyroglutamylglutamylprolineamide, a prostatic, TRH-related tripeptide, on mouse sperm capacitation and fertilizing ability in vitro.

Pyroglutamylglutamylprolineamide, a prostatic tripeptide with structural similarities to thyrotrophin-releasing hormone (TRH), has been found in the seminal plasma of several mammalian species, suggestive of a biological function relating to spermatozoa. Using chlortetracycline (CTC) fluorescence analysis and in vitro fertilization, we have obtained evidence that the tripeptide stimulates mouse sperm capacitation and fertilizing ability in vitro. The tripeptide at concentrations from 5-500 nM was added to sperm suspensions and cells were assessed with CTC after 40 min, insufficient time for complete capacitation by a majority of spermatozoa under standard conditions of incubation. Concentrations of 25 nM and higher significantly promoted capacitation, as evidenced by a decrease in the proportion of acrosome-intact F pattern spermatozoa, characteristic of uncapacitated cells, and an increase in the proportion of acrosome-intact B pattern spermatozoa, characteristic of uncapacitated cells. However, there was no significant stimulation of acrosomal exocytosis. These results suggested that peptide-treated cells would be more fertile than their untreated counterparts. This was confirmed using in vitro fertilization, where the presence of 100 nM peptide during sperm preincubation and gamete coincubation significantly stimulated fertilizing ability (peptide, 56.5% of oocytes fertilized; controls, 26.5%). Comparison of the prostatic tripeptide and TRH effects on capacitation revealed that TRH at a concentration of 250 nM was as effective as the prostatic tripeptide in promoting the F-->B transition but was less effective or ineffective at lower concentrations. In vitro fertilization assessment of the two peptides, at 100 nM, revealed that only the prostatic tripeptide significantly stimulated fertility. Again, this was consistent with the CTC analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1994

Osmotic effects on ram and human sperm membranes in relation to thawing injury.

The effects of hyposmotic and hyperosmotic stresses on ram and human spermatozoa were examined. Human spermatozoa exhibited a precipitate decline in survival at osmolalities below 90 mOsm caused by cells swelling beyond their maximum volume-to-surface area ratio and lysing. Ram spermatozoa exhibited a progressive decline in cell survival at relatively small hyposmotic stresses before exceeding their maximum volume-to-surface area ratio; this prelytic cell loss could be prevented by decreasing the osmolality in a series of 25-mOsm steps. Repeated hyposmotic stress experiments indicated that cells sensitive to prelytic damage constitute a discrete subpopulation within the ram ejaculate. Spermatozoa of both species were apparently resistant to hyperosmotic stresses; human spermatozoa maintained membrane integrity when subject to stresses up to 2.5 Osm and ram spermatozoa up to 1 Osm. However, ram spermatozoa suffered an almost complete and irreversible loss of motility above 600 mOsm. Spermatozoa of both species exposed to hyperosmotic stress and returned to isosmotic conditions exhibited significant cell damage, although ram spermatozoa were the more vulnerable. These observations are related to cryopreservation of spermatozoa.

Animals↗
PubMed1994

Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma.

During incubation of ram spermatozoa at 1 x 10(7) cells mL-1 or less in a simple HEPES-buffered saline medium, high levels of cell death were detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 degrees C. Because the conditions of incubation were similar to those encountered during manipulations for in vitro fertilization, this phenomenon was investigated further. If ram spermatozoa were diluted in an equivalent sucrose-based medium, or if the saline medium was supplemented with 10% seminal plasma, survival was greatly improved (only 5-15% died during a 3-h incubation at 37 degrees C); the protective effect of seminal plasma resided in a 5-10 kDa fraction. Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL-1 whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma. The presence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the saline medium (to render it similar to Tyrode's medium), cell survival was greatly improved (12% died during the 3-h incubation).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Calculated optimal cooling rates for ram and human sperm cryopreservation fail to conform with empirical observations.

The permeability coefficient to water (Lp) and its associated activation energy (Ea) were measured for ram (8.47 microns/min/atm at 25 degrees C, 1.06 kcal/mol) and human (2.89 microns/min/atm at 30 degrees C, 1.93 kcal/mol) spermatozoa. By use of these figures, predictive water loss curves were calculated, from published equations, for different cooling rates from 100 degrees C/min to 100,000 degrees C/min. The calculated curves show that ram spermatozoa cooled at even the fastest rate would be in osmotic equilibrium by -20 degrees C, and human spermatozoa cooled at rates up to 10,000 degrees C/min would be in equilibrium by -15 degrees C. If the nucleation temperature for spermatozoa is taken to be between -20 degrees C and -30 degrees C, then ram and human spermatozoa cooled at these rates would apparently not exhibit any intracellular freezing. There is a significant discrepancy between these calculated optimal cooling rates and the published empirically derived optimal rates of 50 degrees C/min for ram and 10 degrees C/min for human. The failure of ram and human spermatozoa to conform with the established and previously successful model for prediction of optimal cooling rates suggests that damage sustained at high cooling rates may be unrelated to intracellular ice formation.

Animals↗
PubMed1994

Analysis of cytokine gene expression in Graves' disease and multinodular goiter.

Cytokines have a central role in the generation of an autoimmune response and can directly affect the target organ. In Graves' disease, both the infiltrating mononuclear cells and the thyroid follicular cells produce certain cytokines, but the relative contribution of each is unclear, and there are conflicting data on the exact profile of cytokines expressed within the thyroid. To clarify these issues, we used the method of reverse transcription-polymerase chain reaction amplification to analyze cytokine gene expression by intrathyroidal lymphocytes (ITL) and purified thyroid follicular cells (TFC) from six patients with Graves' disease. All ITL samples were positive for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF alpha) messenger ribonucleic acids (mRNAs). Four samples were positive for IL-2 mRNA, and of these, three were also positive for interferon-gamma (IFN gamma). All TFC samples contained IL-6 and IL-8 mRNAs, even after depletion of CD3-positive T-cells. One TFC sample was additionally positive for IL-10 and TNF alpha mRNAs, and in the case of IL-10, this signal was not eliminated by CD3-positive T-cell depletion. IL-4 was not detected in any sample of ITL, TFC, or whole tissue. Semiquantitative analysis showed that the ITL fraction represented the major source of IL-6, IL-8, and TNF alpha mRNAs. By contrast, only three of five multinodular goiter samples were positive for IL-1 alpha mRNA; of these, two were also positive for IL-6, and 1 was positive for IL-8 mRNA. One multinodular goiter sample was positive for IL-8 mRNA alone, but IL-2, IL-4, IL-10, and TNF alpha mRNAS were not detected. These results suggest that although the TFC themselves may express certain cytokines, the ITL population represents the most important source of cytokine production in Graves' thyroid glands. The presence of IL-2, IFN-gamma, and TNF alpha and the absence of IL-4 mRNA in samples of ITL indicate a pattern of cytokine production that most closely resembles that of the TH1 helper T-cell subset. Given the etiological role of thyroid-stimulating antibodies in Graves' disease, the production of which is likely to depend upon TH2 helper T-cell function, it is perhaps surprising that the TH1 subset appears to predominate. It is possible that IL-10 is important in stimulating intrathyroidal autoantibody production, and this cytokine may also play a role in inhibiting cell-mediated thyroid injury in Graves' disease.

Base Sequence↗
PubMed1993

The potential impact of sperm encapsulation technology on the importance of timing of artificial insemination: a perspective in the light of published work.

To facilitate artificial insemination with encapsulated spermatozoa independently of oestrus, ways must be found to extend the life of spermatozoa at body temperature, and to devise encapsulation procedures which will allow progressive release of viable spermatozoa over several days. In the past, the most common approach to the preservation of semen has been the principle of reduced temperature. However, the research literature presents several promising clues to the resolution of these problems; these are reviewed, and new directions are identified and explored. The potential impact of a successful encapsulation technology is recognized as the driving force for implementing the novel research required.

Animals↗
PubMed1993

Determination of water permeability coefficient for human spermatozoa and its activation energy.

Four experiments were conducted to determine the permeability coefficient of human sperm to water (Lp) and its activation energy (Ea). Critical tonicity (tonicity at which 50% of the cells swell and lyse) was determined by equilibrating sperm to 22 degrees C (experiments 1a and 1b), 30, 22, 8, or 0 degrees C (experiment 2a), and 0, -1, -3, -5, or -7 degrees C (experiment 2b) and then exposing them to various hypotonic media (215-3 mOsm). For Lp determination, sperm were equilibrated to 30, 22, 8, or 0 degrees C (experiment 3a), 8, 0, or -3 degrees C (experiment 3b), and -1, -3, -5, or -7 degrees C (experiment 3c), and then were exposed for increasing times to hypotonic (40 mOsm) media. Activation energies were calculated from the results of the latter experiments (experiment 4). Results indicate a temperature-dependent (p < 0.05) critical tonicity, with sperm exhibiting an increased membrane fragility at 8, 0, and -7 degrees C, relative to 30, 22, -1, -3, or -5 degrees C (67.5 +/- 2.4, [mean +/- SEM], 62.7 +/- 2.3, and 61.9 +/- 3.7 mOsm vs. 57.4 +/- 3.4, 57 +/- 1.2, 54.8 +/- 3.4, 60.1 +/- 5.3, and 59.8 +/- 5.2 mOsm, respectively). Human sperm have an Lp of 2.40 +/- 0.20 microns/min/atm at 22 degrees C and an Ea of 3.92 +/- 0.59 kcal/mol between 30 and -7 degrees C. The Ea for cells incubated at temperatures above 0 degrees C (3.92 kcal/mol) show an apparent discontinuity (p < 0.004) in water permeability in supercooled conditions (7.48 kcal/mol). These data suggest that 1) human sperm have a high Lp and low Ea, relative to other cell types, above 0 degrees C; and 2) this high Lp and its low Ea change significantly below 0 degrees C.

Adult↗
PubMed1993

Hyperosmotic tolerance of human spermatozoa: separate effects of glycerol, sodium chloride, and sucrose on spermolysis.

Hyperosmotic stress, which cells experience during the freezing process, and its release during the warming process are both related to cryoinjury. To define optimal cooling or warming rates and prevent osmotic injury to human sperm, information is required regarding the osmotic tolerance of the cells as a function of 1) time, 2) temperature, 3) type of solute, and 4) solute concentration. Human sperm samples were divided into three aliquots. The aliquots were equilibrated at 0, 8, and 22 degrees C, respectively. Different hyperosmotic solutions were prepared by addition of either a permeating cryoprotective agent (glycerol) or nonpermeating solutes (sucrose, non-ionic; or NaCl, ionic) to isotonic Mann's Ringer solution. Aliquots of the prepared solutions were equilibrated at 0, 8, and 22 degrees C, respectively. A small volume (2.5 microliters) of each sperm aliquot was quickly mixed with 50 microliters of each hyperosmotic solution at the corresponding temperature. After times ranging from 5 s to 5 min, 10 microliters of each hyperosmotic cell suspension was abruptly returned to an isosmotic environment by mixing with 500 microliters of Mann's Ringer solution at the corresponding temperature. The plasma membrane integrity of cells after exposure to hyperosmotic stress and after return to isosmotic conditions was measured by a dual staining (carboxyfluoroscein diacetate and propidium iodide) technique and flow cytometry. The morphology of the treated cells was observed by scanning electron microscopy of freeze-substituted sperm. The results indicate that human spermatozoa exhibited a significant posthypertonic lysis/injury, i.e., loss of membrane integrity, when returned to isosmotic conditions after exposure to hyperosmotic solutions of NaCl or sucrose. The higher the hyperosmolality, the more serious the cell injury. The majority of the cells (> 50%) lost membrane integrity when the osmolality was > or = 2000 mOsm. In contrast, if the sperm were not returned to isosmotic conditions, the majority of the sperm in the hyperosmotic solutions appeared to maintain membrane integrity. For a given higher hyperosmolality (> 1000 mOsm), posthypertonic spermolysis was reduced with a decrease of temperature. Cell survival was also affected by time of cell exposure to hyperosmotic environments before cells were returned to the isotonic condition. The shorter the time, the higher the cell survival. When exposed to hyperosmotic glycerol solutions that were isotonic with respect to electrolytes, few cells lost their membrane integrity if the osmolality of glycerol was < 3000 mOsm. For a fixed high osmolality (> 3000 mOsm), the lower the temperature, the higher the percentage spermolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Body Water↗
PubMed1993

No restriction of intrathyroidal T cell receptor V alpha families in the thyroid of Graves' disease.

Recently it has been reported that the intrathyroidal T cells in Graves' disease display restriction in V alpha T cell receptor (TcR) gene family usage, although this is not found with TcR V beta gene families in the same individuals. We have performed a qualitative analysis of TcR V alpha family usage in 12 patients with Graves' disease by reverse transcription and polymerase chain reaction (PCR) amplification of RNA extracted from isolated, unstimulated intrathyroidal lymphocytes and from snap-frozen whole thyroid specimens. No restriction was observed, with 10-15 V alpha gene families being amplified in all cases. The pattern of usage was similar to that in peripheral blood lymphocytes derived from normal subjects (n = 3) and from patients with Graves' disease (n = 3), as well as that present in the thyroids of patients with non-autoimmune toxic multinodular goitre (n = 4). These results indicated that there is no marked restriction of the unselected intrathyroidal T cell population in patients with Graves' disease who have been treated with antithyroid drugs.

Cell Line↗
PubMed1993

T cell receptor beta chain gene polymorphisms in Graves' disease.

Graves' disease, or subgroups of such patients with thyroglobulin antibodies or HLA-DR3, have been associated with a restriction fragment length polymorphism of the T cell receptor beta chain constant region gene but not all studies are in agreement. We have examined 58 patients with Graves' disease and found no abnormal distribution of this polymorphism compared to 33 controls. There was also no significant association with the presence of thyroglobulin antibodies or HLA-DR3, even when the results were pooled with previously collected data (N = 90 patients with TG antibody results and N = 128 patients with HLA-DR types). We also found no abnormal distribution of three polymorphisms of the T cell receptor beta chain variable region genes, using probes which span over 600 kb of this complex. These results argue against an association of these allelic variations of the T cell receptor beta chain with Graves' disease.

Alleles↗
PubMed1993

Vaginal anatomy of the domestic cat (Felis catus) in relation to copulation and artificial insemination.

In the course of an investigation of artificial insemination (AI) in the domestic cat, the site of deposition of the semen during vulval insemination was questioned. A limited study was carried out to ascertain both the anatomy and distensibility of the vagina and urogenital sinus, and the location of the cervix in relation to the vulva. In live restrained female cats a 4 mm diameter Teflon probe could not be inserted more than 20 mm, and elicited a cry similar to the copulatory vocalization when the resistance was encountered. However, a 1 mm diameter ball-tipped needle was passed to a depth of 40 mm. To confirm these observations, four adult female domestic cats were dissected. The cervix was located approximately 45 mm cranial to the vulva. The urogenital sinus narrowed approximately 17.5 mm from the vulva, preventing the passage of a 4 mm probe, and led to a constricted vagina, which permitted the passage of a 1 mm diameter probe to the depth of the cervix. A radiological investigation of a single anaesthetized cat supported these results. The erect penis of the adult male was measured during sexual arousal in five animals; the mean length and width (+/- SD) was 21.2 +/- 2.2 mm and 5.1 +/- 0.5 mm, respectively. These observations lead us to conclude that the stimulus for vocalization during natural mating is distention of the posterior vagina, where semen is probably deposited. The site of semen deposition is uncertain in several accounts of artificial insemination, as the passage of the insemination device depends on its diameter.

Animals↗
PubMed1992

The ionophore-induced acrosome reaction differs structurally from the spontaneous acrosome reaction.

The ultrastructure of the spontaneous acrosome reaction in ram spermatozoa has been compared with that induced by the ionophore, A23187. The spontaneous event was dependent on incubation for 4 h, on the temperature, and on dilution. Apart from the more rapid occurrence of the ionophore-induced event, the mean diameter and distribution of vesicle size was also different. The ionophore-induced vesicles were larger, more irregular, and heterogeneous in size compared with those occurring in the spontaneous acrosome reaction (average diameter 84 nm vs. 60 nm in the spontaneous acrosome reaction). These observations are interpreted in relation to capacitation.

Acrosome↗
PubMed1992

Catalytic activities of human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) expressed in yeast.

A 1.57kb BamH1 fragment containing a full-length human debrisoquine 4-hydroxylase cytochrome P450 (CYP2D6) cDNA was inserted into the BglII site of the yeast expression plasmid pMA91 and the resulting recombinant plasmid, PELT1, introduced into Saccharomyces cerevisiae strain AH22. Microsomes prepared from AH22/pELT1 cells gave an absorption maximum at 448 nm and a P450 content of 67 +/- 31 pmol/mg of microsomal protein. No P450 was detectable in microsomes prepared from AH22/pMA91 control cells. A western blot of microsomes prepared from yeast transformed with pELT1 were probed with a monoclonal antibody to CYP2D6 and revealed a strong band with a molecular mass consistent with that of CYP2D6 from human liver microsomes. No corresponding band was observed with microsomes from control yeast transformed with pMA91 alone. Microsomes from AH22/pELT cells showed catalytic activity towards metoprolol (alpha-hydroxylation and O-demethylation, 0.17 and 0.78 nmol/mg protein/h, respectively); and towards sparteine (2- and 5-dehydrogenation, 1.82 and 0.59 nmol/mg protein/h, respectively). The inhibition of metoprolol metabolism by quinidine (Qd) was 200 times more potent than that of quinine (Qn), both for alpha-hydroxylation (Qd IC50 = 0.05 microM; Qn IC50 = 4 microM) and O-demethylation (Qd IC50 = 0.05 microM; Qn IC50 = 4 microM). Negligible metabolism of tolbutamide and S-mephenytoin, substrates of the 2C sub-family, and of p-nitrophenol, a substrate of CYP2E1, was detected, although a trace of the N-deethylated metabolite of lignocaine, thought to be metabolised by CYP3A4, was detected with microsomes from CYP2D6-expressing yeast cells. The results indicate that yeast cells containing human CYP2D6 cDNA express a functionally active form of the enzyme, the immunochemical and catalytic properties of which are consistent with those of human liver.

Cytochrome P-450 CYP2D6↗