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R C Richards

Publications and source records attributed to R C Richards.

At least 19 recordsLinked to original sources
PubMed2003

Cloning and characterization of the Atlantic salmon serum lectin, a long-form C-type lectin expressed in kidney.

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.

Amino Acid Sequence↗
PubMed2001

Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar).

Antimicrobial activity was detected in acid extracts of liver, intestine, and stomach of healthy Atlantic salmon (Salmo salar). An antimicrobial protein was isolated from salmon liver using acid extraction followed by ammonium sulfate precipitation, large-scale gel filtration chromatography, reverse-phase HPLC, and size exclusion HPLC. The salmon antimicrobial (SAM) protein was found to have a molecular mass of 20,734 Da by MALDI TOF mass spectrometry. Peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry identified the protein as histone H1. The protein had a minimal inhibitory concentration of 31 microg/mL against E. coli D31 in a plate clearing assay. The effect of the SAM protein on bacterial morphology was indistinguishable from that of (Ala-(8,13,18))-magainin II, as shown by scanning electron microscopy, which suggests that the protein disrupts E. coli membranes in a manner similar to that of most antimicrobial peptides. This protein may act as an antimicrobial in vivo through active secretion or by release from cells during infection-related apoptosis.

Animals↗
PubMed2001

Cloning of glycerol-3-phosphate dehydrogenase cDNAs from two fish species and effect of temperature on enzyme expression in rainbow smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) can accumulate extreme levels of glycerol in their blood during winter. Low temperatures are required for glycerol accumulation in smelt blood and the enzyme glycerol-3-phosphate dehydrogenase (GPDH) has been suggested to play a role in glycerol production/concentration in this species. In the present study, cDNA sequences encoding glycerol-3-phosphate dehydrogenase (GPDH) from rainbow smelt and Atlantic salmon (Salmo salar) were cloned. The encoded GPDH protein sequences were very similar to one another (88% identity). Using RT-PCR, GPDH mRNA was detected in skin, gill, heart, head kidney, brain and liver from both salmon and smelt obtained in December. However, GPDH was not detected in salmon intestine and spleen or in smelt intestine. Examination of GPDH expression in smelt liver during February by Northern blotting revealed temperature regulation. Elevation of the temperature resulted in a significant decrease in liver GPDH transcript level. Serum glycerol levels decreased concomitantly. These findings suggest a role for GPDH in the accumulation of glycerol in smelt at low temperatures.

Amino Acid Sequence↗
PubMed1995

Total number of immunocompetent cells in the normal rat epididymis and after vasectomy.

The aim of this study was to make unbiased quantitative estimates of the number of T8 cytotoxic cells and T4 helper cells and macrophages in the head and tail of rat epididymides, using newly developed design-based stereological methods in fertile control animals, and to compare these with results from vasectomized animals. The location of these cells within the intra- and interepithelial compartments of the epididymis was also studied. In control animals, both the T4 helper cells and macrophages and T8 cytotoxic cells were found in significantly higher numbers (P < 0.01) in the head interepithelial location than in the intraepithelial site. In addition, there were significantly more (P < 0.01) T8 cytotoxic cells in the tail interepithelial region. Within the head region of vasectomized rats, there was a significant increase in T8 cytotoxic cells in the intraepithelial location (P < 0.01), and a decrease in T4 helper cells and macrophages in the interepithelial region (P < 0.05). Serum samples from vasectomized animals, when compared with those from control animals, showed a significant increase in cytotoxic antisperm antibody. This suggests that, in response to the increased spermatozoal antigenic absorption that takes place in the epididymis after vasectomy, the immune system redistributes its T cell subsets to modify the immune tolerance that may occur.

Animals↗
PubMed1994

Localization of EGF receptors in frozen tissue sections by antibody and biotinylated EGF-based techniques.

We developed a sensitive EGF receptor detection method for frozen tissue sections using biotinylated EGF as the primary reagent. The method was directly compared with an immunohistochemical technique based on an anti-EGF receptor monoclonal antibody (MAb EGFR1) in normal human and rat tissues and in human tumors. The method was more sensitive than a previously published biotinylated EGF-based technique. In normal human tissues and in 37 of the 50 tumors, the binding pattern mirrored that of positive staining with EGFR1. Five further tumors showed weak immunoreactivity, but in these no binding of biotinylated EGF was detected. The remaining eight tumors were negative by both techniques. The discordant cases may reflect a lower level of sensitivity of the ligand-binding technique or, alternatively, abnormal receptors may have been expressed in these tissues. EGF receptors could be detected in rat liver with biotinylated EGF but not with the antibody, indicating the usefulness of the ligand-based technique in cross-species studies.

Animals↗
PubMed1991

The effects of intracolonic EGF on mucosal growth and experimental carcinogenesis.

Although intra-luminal epidermal growth factor (EGF) may stimulate cell proliferation in the upper gastrointestinal tract, its role in the large bowel has not been established. We have therefore studied the effect of intra-rectal EGF administration on both normal growth and carcinogenesis in the rat colon. Colonic cancer was induced in rats with azoxymethane (10 mg kg-1 week-1 for 12 weeks s.c.) and controls dosed with saline. In each group, animals were randomised to receive EGF (12 nM, 0.8 nM or saline control) in 0.5 ml saline via a rectal tube daily for 24 weeks. At this time, crypt cell production rates (CCPRs) were determined at two sites in the colon: one of maximal and another of minimal exposure to EGF (5 cm and 10 cm from the anal margin respectively). No effects of EGF were seen at 10 cm. The lower dose of EGF gave CCPRs that mirrored the control values. The higher dose of EGF in the animals not treated with azoxymethane stimulated mucosal growth. Azoxymethane increased in CCPR, but this was suppressed by the high dose of EGF. These results suggest that (1) luminal EGF and azoxymethane independently increase the colonic CCPR and their combined effect is not synergistic but antagonistic; (2) EGF may have a role in normal epithelial growth, but does not potentiate colonic carcinogenesis in this model.

Animals↗
PubMed1988

Epidermal growth factor (urogastrone) in human seminal plasma from fertile and infertile males.

Epidermal growth factor/urogastrone (EGF/URO) was measured in the seminal plasma of 214 untreated patients attending an infertility clinic. Patients were assigned groups according to sperm density and progressive motility and in the case of azoospermia into an obstructive etiology or germinal failure. There was no significant variation in mean EGF/URO levels between groups, suggesting that this peptide plays no role in the density or motility of sperm associated with fertility. Other functions this peptide may fulfill in the female reproductive tract, including that of immunosuppression, are discussed.

Epidermal Growth Factor↗
PubMed1987

Immunocytochemical localisation of the antibody which breaches the blood-testis barrier in sympathetic orchiopathia.

The phenomenon of sympathetic orchiopathia, in which ischaemia of one testis results in histological damage to the contralateral testis, is now well documented. This study used immunocytochemical techniques to investigate whether this damage is caused by antibody penetrating the "blood-testis" barrier and affecting spermatogenic and supporting cells of the seminiferous tubules. It has been demonstrated that IgM antibodies breach the "blood-testis" barrier of the contralateral testis 7 days after infarction and that they are replaced by IgG antibodies after 28 days. Cells affected are the epithelial cells of the membrana propria, the Sertoli cells and immature spermatogenic cells. Beyond 28 days after infarction no significant amounts of antibody of either class were demonstrated within the seminiferous tubules of contralateral testes.

Animals↗
PubMed1987

Uptake and metabolism of epidermal growth factor in the perfused human placenta.

Uptake of 125I-labelled epidermal growth factor into trophoblast, and its subsequent fate, was studied in an isolated dually-perfused lobule of term human placenta. 125I-EGF added into the maternal circulation was rapidly taken up into the placental tissue where a portion was degraded and most of the breakdown products released back into the maternal circuit. At the end of the 2 h perfusion, radioactivity in the tissue accounted for 52% of the initial dose. 12.9% of the radioactivity remaining in the maternal circuit at the end of the perfusion, amounting to only 5.2% of the initial activity, could be identified as intact EGF by immunoaffinity chromatography. About 45 min after the start of the perfusion there was a sustained rise in the 125I activity in the fetal circulation accounting for 4.6% of the initial activity, and a small proportion of this (0.22% of the dose) could be immunologically characterised as EGF. In the presence of the acidotrophic agent chloroquine, there was a similar rapid clearance from the maternal circulation, which was not associated with breakdown. The tissue retention was slightly enhanced and there was very little transfer of activity into the fetal circulation.

Biological Transport↗
PubMed1986

Assembly and characterisation of a multi-component cytoskeletal gel from adrenal medulla.

Incubation of bovine adrenal medullary cytoplasmic extracts results in the formation of three-dimensional supramolecular gels. Ultrastructurally, the gels display a network of fibres similar in appearance to the cytoskeleton within intact chromaffin cells. Analysis of the protein composition using both electrophoretic and immunoblotting techniques indicates that the gels are composed exclusively of cytoskeletal elements; microfilaments, microtubules and intermediate filament proteins have been identified as having a number of actin-associated proteins. Among the latter class of components the following polypeptides have been identified: filamin (300 kDa), fodrin (240 kDa), a 235 kDa polypeptide, myosin (200 kDa), caldesmon (70 kDa) and tropomyosins (39 kDa). All of these polypeptides co-sedimented with F-actin when gels were assembled in the absence of Ca2+. When gelation was performed in the presence of 10 microM Ca2+ actin, the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosin were all absent from the gels. These results may suggest that the 235 kDa polypeptide, 70 kDa caldesmon and tropomyosins could act either individually or as a functional regulatory unit in controlling the Ca2+-activated reorganisation of the actin network in the cytoplasmic gels.

Actins↗
PubMed1986

Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and pHi.

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), at nanomolar concentrations, induces rapid (t1/2 approximately 30 s) protrusion of multiple petal-shaped lamellae by neutrophil leucocytes. Lamellae are richly endowed with actin filaments as determined by the localization of rhodamine-phalloidin, suggesting extensive assembly at the cell cortex. Direct measurement of the proportion of total cell actin which is polymerized, by using a deoxyribonuclease I inhibition assay, indicates that the proportion of polymerized actin approximately doubles, and that assembly initiated by 30 nM TPA occurs with no obvious lag phase and with a t1/2 of about 30 s. A half-maximal response was induced at 2 nM TPA. Since both actin assembly and protrusion of lamellae are completely inhibited by 10(-6) M cytochalasin D, protrusion of lamellae is presumably dependent on actin filament assembly. To examine whether TPA induces actin assembly via changes in [Ca2+]i or pHi, these parameters were monitored in cells loaded with the fluorescent indicators quin2 and quene1 respectively. Addition of TPA caused no change in [Ca2+]i but a biphasic change in pHi. To examine further the potential role of ionic changes in regulation of actin assembly, the morphological responses of cells to TPA were monitored in severely Ca2+-depleted cells, or cells in which pHi had been experimentally raised or lowered by simultaneous additions of a weak base (NH4Cl) or weak acid (CH3COONa) respectively. The protrusion of lamellae induced by TPA was completely unaffected by these experimental manipulations indicating that TPA can directly regulate actin assembly, and hence morphology, by mechanisms essentially independent of [Ca2+]i and pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Urogastrone-epidermal growth factor concentrations in prostatic fluid of normal individuals and patients with benign prostatic hypertrophy.

High concentrations (272 +/- 33 ng/ml) of urogastrone-epidermal growth factor were measured in prostatic fluid from normal males by a specific radioimmunoassay. Significantly lower concentrations (155 +/- 24 ng/ml) were observed in the prostatic fluid of patients with benign prostatic hypertrophy than in the age-matched normal controls (2P less than 0.01). The growth factor content of seminal fluid was accounted for by the contribution of prostatic fluid. Immunochemical studies failed to show evidence of synthesis within the gland nor could high affinity receptors for the protein be demonstrated in membrane preparations of the gland.

Chromatography, Gel↗
PubMed1984

Prolactin concentrations in mouse milk during lactation.

Prolactin levels in mouse milk increased from the day of parturition to give a concentration of 230 ng/ml on days 2 and 3 of lactation. Thereafter, levels dropped to 140 ng/ml by day 6 and were maintained at this concentration until weaning.

Animals↗
PubMed1984

The use of trypsin to improve the localization of immunoglobulins in semi-thin frozen sections of the mammary gland.

Frozen sections, 0.5 micron thick, of the lactating mouse mammary gland have been used to localize immunoglobulins A and G and serum albumin throughout the connective tissue stroma, in the lumina of blood vessels, in milk stored in the alveoli and in the lateral spaces between adjacent epithelial cells. In addition, the immunoglobulins were localized to their specific plasma cells in the connective tissue stroma. Serum albumin was further identified within the mammary epithelial cells as small spots of fluorescence scattered throughout the cytoplasm. The immunoglobulins were not localized within these cells in untreated sections, but in sections treated with trypsin and Soybean trypsin inhibitor, it was possible to identify a similar distribution to that for serum albumin. The spots of fluorescence representing the intracellular localization of the immunoglobulins and serum albumin were frequently found in association with the periphery of intracellular lipid droplets.

Animals↗
PubMed1984

Fc receptor-directed phagocytic stimuli induce transient actin assembly at an early stage of phagocytosis in neutrophil leukocytes.

The phagocytic pathway for the uptake of multivalent Fc complexes by neutrophil leukocytes can be partially dissected by using a pulse-chase protocol at different chase temperatures. Utilising a deoxyribonuclease I inhibition assay, we show that an early response to recognition of phagocytic substrate by neutrophils is a rapid (less than 10 sec) polymerization of actin. Net actin assembly is transient and is reversed at higher chase temperatures which appears to allow for further progress of the pulse of phagocytic substrate along the pathway. Assembly of actin monitored in whole cells is paralleled by an increase in total actin retained in the cortical filamentous network of detergent-insoluble ghosts prepared from phagocytosing neutrophils. These data provide direct evidence for the reversible reorganisation of actin filaments in the cortical cytoplasm of phagocytic cells during phagocytosis. Furthermore, this experimental system provides an opportunity to study the characteristics of ligand-induced actin assembly, and subsequent disassembly in situ.

Actins↗
PubMed1983

Urogastrone and lactose concentrations in precolostrum, colostrum, and milk.

The concentration of urogastrone (URO) and lactose was measured in breast secretions from four women. URO levels were high in precolostrum and fell markedly around the time of parturition to relatively constant low levels in mature milk. When milk samples were fractionated on a Biogel P10 column, the major peak of immunoreactivity occurred at the point at which pure URO eluted. There was good correspondence between the fall in URO levels at birth and the increase in lactose levels, suggesting that the decrease in URO was a dilution effect as the osmotic influence of lactose increased.

Colostrum↗