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Biomedical subjects

R M Hill

Publications and source records attributed to R M Hill.

At least 19 recordsLinked to original sources
PubMed2002

Characterisation of two serine protease inhibitors expressed in the pituitary gland.

Serine protease inhibitors (serpins) are a family of structurally related proteins that play key roles in the regulation of proteolytic homeostasis. We have isolated a novel intracellular serpin, termed raPIT5a, from the rat pituitary gland. Northern blot analysis indicated raPIT5a mRNA expression in a range of tissues, including the adrenal gland and the brain. In situ hybridisation histochemistry revealed raPIT5a mRNA expression in specific cell populations in the rat pituitary gland, adrenal gland, and pancreas. Based on sequence similarities to other intracellular serpins, we predicted raPIT5a may inhibit the pro-apoptotic serine protease granzyme B. We confirmed this experimentally by identification of a stable inhibitory complex between granzyme B and raPIT5a. To determine whether granzyme B or granzyme B-related enzymes were expressed in the rat pituitary gland, we performed PCR using primers predicted to amplify granzyme B and two other published granzyme sequences. We identified rat natural killer protease-1 (RNKP-1), the rat homologue of granzyme B, and a novel putative serine protease highly similar to granzyme-like protein III (GLP III), which we termed GLP IIIa. These data suggest raPIT5a may regulate apoptosis in the pituitary by inhibition of granzyme B or GLP IIIa, or members of the caspase enzyme family which have similar substrate specificity. We have also identified expression of a second serpin, called neuroserpin, in pituitary tissue and found that it alters the morphology of the AtT20 corticotrope cell line, presumably through changes in cell adhesion. These results identify new roles for serpins in pituitary cell function.

Amino Acid Sequence↗
PubMed2001

A quantified analysis of the dielectric dispersion in a simple emulsion system.

The dielectric response of a simple, self emulsifying, system, based on a polysorbate and a medium chain length triglyceride, has been investigated as a function of the water content in the range 0 to 80% by weight. The low frequency capacitive dispersions have been characterised in terms of an electrode barrier layer dispersion, and the bulk conductance and capacitance have been quantified. It is shown that the barrier parameters are essentially insensitive to the water content, over concentrations from 2.5 to 75% by weight, and that the magnitude of the bulk emulsion conductance determines the form of the dispersions which can be observed.

Chemical Phenomena↗
PubMed2001

Expression, purification, and functional characterization of the serine protease inhibitor neuroserpin expressed in Drosophila S2 cells.

Neuroserpin (NS) is a serine protease inhibitor (or serpin) that is widely expressed in the developing and adult nervous systems. It has been implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration, and axogenesis. To aid in the characterization of this new serpin we have established a high-level expression system in Drosophila S2 cells and developed a purification strategy to obtain neuroserpin for functional studies. Suspension cultures of S2-NS cells secreted recombinant neuroserpin into the medium. High-level expression was maintained when the cells were switched to a nonselection serum-free medium for 3-4 days to facilitate protein purification. Recombinant neuroserpin was purified by sequential chromatography on Macroprep ceramic hydroxyapatite, Type I, POROS HQ20, Resource Q, and Superdex 75 HR 10/30 media. Two secreted forms of neuroserpin were observed with molecular weights of approximately 49 and approximately 50 kDa which may represent alternative glycosylation at three putative N-linked glycosylation sites. Amino acid sequence analysis indicated three NH(2)-terminal sequences. The major sequence was generated by cleavage at the Gly(18)-Ala(19) bond consistent with removal of an 18-amino-acid signal peptide. Two further sequences were identified each with one fewer amino acids at the NH(2)-terminus. All three NH(2)-terminal sequences were also identified by mass spectrometric analysis of neuroserpin following trypsin digestion. Mass spectrometry also confirmed the protein had an intact carboxyl terminus while complex formation assays indicated the inhibitor was functionally active. In summary, Drosophila S2 cells offered a nonlytic stable expression system for the continual production of neuroserpin in high-density suspension cultures.

Amino Acid Sequence↗
PubMed2001

Fractures of the pubic rami. Epidemiology and five-year survival.

We reviewed 286 consecutive patients with a fracture of a pubic ramus. The overall incidence was 6.9/100,000/year in the total population and 25.6/100,000/year in individuals aged over 60 years. The mean age of the patients was 74.7 years and 24.5% suffered from dementia. Women were affected 4.2 times more often than men. After injury, geriatric rehabilitation was frequently required and although most surviving patients returned to their original place of residence, their level of mobility was often worse. The overall survival rates at one and five years were 86.7% and 45.6%, respectively. Multiple logistic regression analysis showed that age and dementia were the only independent significant factors to be predictive of mortality (p < 0.05). Patients with a fracture of a pubic ramus had a significantly worse survival than an age-matched cohort from the general population (log-rank test, p < 0.001), but this was better than patients with a fracture of the hip during the first year after injury, although their subsequent mortality was higher. Five years after the fracture there was no significant difference in survival between the two groups.

Adolescent↗
PubMed2000

Neuroserpin is expressed in the pituitary and adrenal glands and induces the extension of neurite-like processes in AtT-20 cells.

Two cDNAs encoding the serine protease inhibitor (serpin) neuroserpin were cloned from a rat pituitary cDNA library (rNS-1, 2922 bp; rNS-2, 1599 bp). In situ hybridization histochemistry showed neuroserpin transcripts in the intermediate, anterior and posterior lobes of the pituitary gland and medullary cells in the adrenal gland. Expression of rNS-1 mRNA was restricted to selected cells in the pituitary gland. Analysis of purified secretory-granule fractions from pituitary and adrenal tissues indicated that neuroserpin was found in dense-cored secretory granules. This result suggested that endocrine neuroserpin may regulate intragranular proteases or inhibit enzymes following regulated secretion. To investigate the function of neuroserpin in endocrine tissues we established stable anterior pituitary AtT-20 cell lines expressing neuroserpin. Cells with increased levels of neuroserpin responded by extending neurite-like processes. Extracellular proteolysis by serine protease plasminogen activators has been suggested to regulate neurite outgrowth. As neuroserpin inhibits tissue plasminogen activator (tPA) in vitro, we measured plasminogen-activator levels. Zymographic analysis indicated that AtT-20 cells synthesized and secreted a plasminogen activator identical in size to tPA. A higher-molecular-mass tPA-neuroserpin complex was also observed in AtT-20-cell conditioned culture medium. tPA levels were similar in parent AtT-20 cells and a stable cell line with increased levels of neuroserpin. There was no accumulation of a tPA-neuroserpin complex. Together these results identify endocrine cells as an important source of neuroserpin. Moreover they suggest that neuroserpin is released from dense-cored secretory granules to regulate cell-extracellular matrix interactions through a mechanism that may not directly involve tPA.

Adrenal Glands↗
PubMed2000

Oxygen: how well is the closed eye being served?

BACKGROUND: While the environment of the naturally closed eye substantially challenges the oxygen-dependent processes of the cornea, the presence of a contact lens further exacerbates those stresses. The purposes of this study are: (1) to describe responses of the human cornea under closed eye conditions to a wide range of Dk/t environments as ratios of their corresponding normal, open-eye (physiological) baseline rates; (2) to describe the difference ("hypoxic gap") function that separates those closed-eye responses from parallel responses for the open eye; and (3) to present predictive models for all three (closed-eye, open-eye, and difference) response functions in both graphical and mathematical forms. METHODS: Oxygen uptake rates were measured polarographically: for the normal open eye and after both closed- and open-eye wear of six rigid contact lenses of transmissibilities ranging from 0 to 91 x 10(-9) (cm/sec)(ml O2/ml mmHg). RESULTS: Responses from the closed-eye and open-eye series to those six Dk/t levels were compared both graphically and statistically. Those response series were described by best-fit equations, from which a model for Dk/t effectiveness in meeting corneal oxygen needs under both closed- and open-eye conditions was developed. CONCLUSIONS: Response series for both closed and open eyes were found to be well described by natural log equations (R2 values > 0.99). Statistically, separation of those two series could be demonstrated (p < 0.05) by the Dk/t level of 12.8 x 10(-9) (cm/sec)(ml O2/ml mmHg). While, by the highest Dk/t level observed here of 91 x 10(-9) (cm/sec)(ml O2/ml mmHg), corneal responses for the open eye have already moderated down to just 1.5x the "ideal standard," closed-eye response rates are still averaging 2.7x that baseline. The difference ("hypoxic gap") function remaining between those closed- and open-eye curves appears to be stabilizing in its magnitude by 91 x 10(-9) (cm/sec)(ml O2/ml mmHg) as well.

Adult↗
PubMed2000

The thickness of the human precorneal tear film: evidence from reflection spectra.

PURPOSE: Interferometric methods have considerable potential for studying the thickness of layers of the human tear film and cornea because of their ability to make noninvasive, accurate, and rapid measurements. However, previous interferometric studies by Prydal and Danjo yielded tear thickness values near 40 and 11 microm, respectively, considerably greater than estimates made by invasive methods of 4 to 8 microm. Using a modified version of Danjo's method, interference effects from the tear film and cornea were studied, with the aim of correlation with known structure and optical properties of the cornea and hence determining the most probable value of tear film thickness. METHODS: Reflectance spectra from the human cornea were measured at normal incidence. These spectra show oscillations whose maxima correspond to constructive interference between light reflected from the air surface and from some deeper surface. The frequency of these spectral oscillations is proportional to the thickness of the layer between the air surface and the second surface. Therefore, Fourier analysis of reflectance spectra can be used to determine the thickness of layers of the tear film and cornea. In the main experiment, 36 low-resolution spectra were obtained from six normal eyes for measuring thickness up to 100 microm. Control experiments included measurements of the time course of thickness changes and high-resolution spectra for measuring thickness up to 1000 microm. RESULTS: For the main experiment, in the thickness range 1 to 100 microm, the strongest peak in the Fourier transform was near 3 microm (range, 1.5-4.7 microm) beneath the air surface. In the range 20 to 100 microm, the strongest peak was near 55 microm (range, 50-59 microm) for all 36 spectra; none were in Prydal's range near 40 microm. This 55-microm peak is consistent with a reflection from the basement membrane of the epithelium. Time course measurements after a blink show that the 3-microm peak is not an artifact. High-resolution spectra gave a peak near 510 microm, corresponding to the complete thickness of the cornea (plus tear film). This peak had a contrast similar to that of the 3-microm peak. CONCLUSIONS: These studies did not confirm Prydal's estimate of approximately 40 microm. Nor were there prominent peaks near Danjo's value of approximately 11 microm, except in cases of probable reflex tears. Because the reflection at the aqueous-mucus boundary would be expected to be weaker than that from the epithelial surface, the 3-microm peak is unlikely to correspond to the aqueous layer (rather than the complete tear film). The proposal that the 3-microm peak corresponds to a reflection from the front of the cornea is supported by the demonstration of a peak of similar contrast from the back of the cornea. Thus, the current evidence consistently supports a value of approximately 3 microm for the thickness of the human precorneal tear film.

Adult↗
PubMed1999

The "physiologically effective" correlates: peDk/L and peDk.

Introduced here are two predictive parameters, peDk/L and peDk. The prefix pe- (for physiologically effective) indicates that such values are estimates of lens transmissibility (Dk/L) and material permeability (Dk) and have been derived through a reverse application of the Residual Hypoxia model of Smith, relating corneal response (in hypoxic stress units) to lens transmissibility (Dk/L). Here, using independent databases, physiological responses to simple oxygen pathways (one layer, one material) but of unknown Dk value were entered into that model to estimate the Dk/L and (then by calculation) the Dk value of single vision rigid gas permeable (RGP) contact lenses. Also demonstrated was the application of this strategy for estimation of composite (integrated) Dk/L and Dk values of complex (multi-layer, multi-material) oxygen pathways, wherein one or more material Dk values and/or one or more local thickness are unknown.

Contact Lenses↗
PubMed1999

What is the oxygen environment under an encapsulated segment bifocal RGP contact lens?

BACKGROUND: A bifocal rigid gas permeable (RGP) contact lens is now available that totally encapsulates an add segment of a different material within its inferior aspect. The purposes of this study were to determine the effects of the major lens and of its encapsulated segment region on the oxygen uptake rates of underlying cornea. METHODS: Corneal oxygen uptake rates of 10 human corneas were measured--both centrally and inferiorly--for non-wearing conditions and then immediately after 300-second wearing periods of a polymethylmethacrylate (PMMA) lens and of this RGP-encapsulated segment bifocal contact lens. Corneal responses associated with these three environmental conditions were compared statistically (i.e., without the influence of blink-driven tear flow). RESULTS: Under the nonblinking conditions maintained (i.e., with oxygen availability restricted to lens transmissibility alone), central corneal hypoxia was found for the RGP bifocal lens to be only 47% of that induced by the PMMA (Dk/L = 0) condition, while the cornea under the encapsulated segment of the bifocal lens averaged 78% of the hypoxic response induced by that same condition. Central site responses were found to be significantly different for the bifocal and the PMMA lens (p < 0.001), as they were for the two inferior sites with these two lenses (p < 0.001). All lens-related responses were found to be significantly different from the no lens condition (p < 0.001). CONCLUSIONS: Using Smith's model (JAOA, 1997) to estimate lens transmissibility, the major lens of this RGP bifocal design appears to meet approximately 80% of the Holden-Mertz daily-wear oxygen requirement--even without post-lens tear exchange, but only about 25% of that requirement appears to be met beneath the encapsulated segment region. Efficient supplementary infusion of oxygen under this lens by blink-driven tear exchange, then, is necessary to maintain optimal corneal health.

Adult↗
PubMed1998

A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B.

We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374-amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS-stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.

Amino Acid Sequence↗
PubMed1998

Long-term neurodevelopmental outcome after intrauterine transfusion for the treatment of fetal hemolytic disease.

OBJECTIVE: The aim of the study was to assess the developmental outcome of neonatal survivors of hemolytic disease of the neonate treated with modern intrauterine transfusion techniques. STUDY DESIGN: In this prospective, observational study, auditory evoked-response tests were performed in the nursery. Neurodevelopmental evaluation with the Gesell Developmental Schedules was performed between 9 and 18 months of corrected age to assess motor skills, language development, comprehension capacity, and social skills. The McCarthy Scales of Children's Abilities were administered between 36 and 62 months. RESULTS: Forty children who survived severe fetal hemolytic disease were followed up until 62 months old. Demographic data included gestational age at first intrauterine transfusion (26.4 +/- 3.7 weeks), median number of intrauterine transfusions (4, range 1-8), lowest fetal hematocrit (20.2% +/- 7.8%), peak fetal bilirubin (7.1 +/- 2.1 mg/dL), incidence of hydrops fetalis (45%), and mean gestational age at delivery (35.6 +/- 2.2 weeks). One case of severe bilateral deafness and 1 case of right spastic hemiplegia were diagnosed. The Gesell Developmental Schedules score was assessed between 9 and 18 months of corrected age in 22 infants. The global developmental quotient was 101.9 +/- 9.5 (mean for normal population is 100). Regression analysis revealed no correlation between the global developmental quotient and gestational age at the first intrauterine transfusion, gestational age at birth, or the severity of the fetal hemolytic disease (fetal hematocrit, fetal bilirubin, presence of hydrops fetalis, total number of intrauterine transfusions, duration of neonatal phototherapy, and number of neonatal exchange transfusions). Eleven of the 40 children were followed up until they were 62 months old, and the McCarthy Scales of Children's Abilities were administered. The mean cognitive index was 107.6 +/- 9.4 (90-109 is considered average). CONCLUSION: Despite severe fetal hemolytic disease, normal developmental outcome can be expected for children treated with intrauterine transfusions.

Bilirubin↗
PubMed1998

Effects of rigid contact lens prism ballasting on oxygen access to the cornea under static and dynamic conditions.

The effects of rigid contact lens material [polymethyl methyacrylate (PMMA) and itabisfluorofocon A] and prism ballasting (0, 1.5, 2.25, and 3 delta) on tear pump efficiency were studied by measuring corneal oxygen uptake rates on the right eyes of six human subjects under three conditions: (1) normal open eye; (2) after 5 min of static (without blinking) wear of the contact lens; and (3) after 5 min of dynamic (with blinking once every 5 s) wear of the same lens. As expected, corneal oxygen uptake rates (i.e., demand for oxygen) measured with the itabisfluorofocon A material were significantly lower (p < 0.0001) than those measured with PMMA under both static and dynamic conditions. Under static conditions, no significant differences were found across materials among the corneal oxygen uptake rates associated with the four amounts of prism ballasting (p = 0.0514). However, under dynamic conditions, significant differences (p < 0.0001) were found across materials among corneal oxygen uptake rates associated with the four amounts of prism ballasting, with the lower amounts of prism ballasting being associated with lower corneal oxygen uptake rates. The changes in the measured corneal oxygen uptake rates from static to dynamic conditions, relative to those measured for the normal open eye, served as an index of tear pump efficiency. There were no significant differences in these changes for the prism amounts studied; however, static condition data were significantly higher than dynamic condition data for the 0 delta and 1.5 delta lenses only, whereas greater amounts of prism ballasting resulted in no reduction in oxygen uptake under dynamic conditions. In addition, significantly greater differences between static and dynamic condition data were found for the PMMA material than for itabisfluorofocon A.

Adult↗
PubMed1997

Rigid lens dynamics: lid effects.

PURPOSE: Lid architecture, lid tension, blink action, and blink rate may all influence rigid lens centration and stability. The aims of this study were to assess the nature of the relationship between lid geometry and lens position and to examine the influence of lens-lid interactions on the association between lens position and lens center of gravity and mass. METHODS: Eight subjects (four with high riding lenses and four with low riding lenses) participated in the study. Each subject was fit with 12 lenses-six designs in each of two materials. Lens center of gravity was calculated and lens mass was measured in every case. For each lens, the following four lens dynamics variables were assessed over a five blink cycle for both the vertical and horizontal meridians: 1) initial lens position; 2) settled lens position; 3) amount of lens movement; and 4) rate of lens movement. Lower lid position and palpebral aperture height were measured for each subject. RESULTS: Graphical analysis showed that a low upper lid position and small palpebral aperture promoted superior lens decentration, while a high upper lid position and wide palpebral aperature predisposed an individual to inferior lens decentration. Significant lid-lens overlap was a common finding with high riding lenses in both the initial and settled lens positions. The results also suggested that while a rigid lens tended to become more stable as the lens center of gravity shifted further behind the corneal apex, this gravitational effect was reduced for lenses that interacted with the upper lid. CONCLUSIONS: Lid geometry can influence rigid lens centration and stability by modifying the effects of lens design and lens mass. We recommend that consideration be given to the nature of potential lens-lid interactions prior to lens fitting.

Blinking↗
PubMed1997

Corneal responses to lens transmissibility.

BACKGROUND: Lens transmissibility (Dk/L) and corneal relief from hypoxia are not linearly related. We describe graphically and by equations the relationship between corneal relief (scaled in oxygen shortfall units) and Dk/L across the transmissibility range of 1.2 to 189 x 10(-9) (cm/sec)(ml O2/ml mmHg). METHODS: Corneal responses to 28 Dk/L levels across the transmissibility range were expressed in oxygen shortfall units (osús), a relative scale of 100 steps bounded by each cornea's normal oxygen uptake rate with no contact lens in place (set = to 0 osús), and its maximum uptake rate for the open eye due to static PMMA lens wear (set = to 100), thus allowing the degree of hypoxic stress exhibited by all corneas to be compared on the same relative scale. RESULTS: Responses of 11 human corneas to each of those 28 Dk/L steps were recorded across that entire transmissibility range, and their collective relationship described graphically. CONCLUSION: The graphic composite of all corneal responses to transmissibility steps across the Dk/L range of 1.2 to 189 X 10(-9) (cm/sec)(ml O2/ml mmHg) was found to be monotonic and continuous. While no single equation was found that optimally described (predicted) corneal responses across that entire spectrum, equations of the quadratic form fit limited domains of Dk/L (r2 values exceeding 90%) adequately for most laboratory and clinical applications.

Adult↗
PubMed1996

Contact lens transmissibility: effects on delivery of oxygen to the cornea.

The purpose of this study was to determine the effects of rigid contact lens materials of various permeabilities and identical design on the oxygen shortfall of the human cornea and to explore differences in oxygen delivery to the cornea associated with contact lens materials of different modulus of elasticity values. Measurement of corneal oxygen uptake was performed for six human subjects under three conditions: (1) normal open eye; (2) after the static wear of six materials; and (3) after the dynamic wear of the same six materials. The 6 materials had Dk values that ranged from 0.02 to 127 (cm2/s)(ml O2/ml x mm Hg) and modulus of elasticity values from 1300 to 2200 MPa. Major results include: (1) that the oxygen shortfall of the cornea decreased with increasing lens transmissibility under both static and dynamic conditions, with significant differences (p < 0.001) being demonstrated among materials; and (2) significantly larger differences (p < 0.001) between the static and dynamic condition data were seen for materials of low Dk but were not predictable by lens modulus (p = 0.43, r2 = 0.018). The corneal demand differences between high and low Dk materials are possibly related to changes in corneal oxygen dynamics rather than to differences in tear exchange.

Adult↗