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Biomedical subjects

M L Robinson

Publications and source records attributed to M L Robinson.

At least 19 recordsLinked to original sources
PubMed1999

A transgenic animal model of osmotic cataract. Part 1: over-expression of bovine Na+/myo-inositol cotransporter in lens fibers.

PURPOSE: Intracellular osmotic stress is believed to be linked to the advancement of diabetic cataract. Although the accumulation of organic osmolytes (myo-inositol, sorbitol, taurine) is thought to protect the lens by maintaining osmotic homeostasis, the physiologic implication of osmotic imbalance (i.e., hyperosmotic stress caused by intracellular over-accumulation of organic osmolytes) on diabetic cataract formation is not clearly understood. Studies from this laboratory have identified several osmotic compensatory mechanisms thought to afford the lens epithelium, but not the lens fibers, protection from water stress during intervals of osmotic crisis. This model is founded on the supposition that the fibers of the lens are comparatively more susceptible to damage by osmotic insult than is the lens epithelium. To test this premise, several transgenic mouse lines were developed that over-express the bovine sodium/myo-inositol cotransporter (bSMIT) gene in lens fiber cells. METHODS: Of the several transgenic mouse lines generated, two, MLR14 and MLR21, were analyzed in detail. Transgenic mRNA expression was analyzed in adult and embryonic transgenic mice by a coupled reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization on embryonic tissue sections, respectively. Intralenticular myo-inositol content from individual mouse lenses was quantified by anion exchange chromatography and pulsed electrochemical detection. Ocular histology of embryonic day 15.5 (E15.5) embryos from both transgenic (TG) families was analyzed and compared to their respective nontransgenic (NTG) littermates. RESULTS: Both RT-PCR and in situ hybridization determined that transgene expression was higher in line MLR21 than in line MLR14. Consistent with this, intralenticular myo-inositol from MLR21 TG mice was markedly higher compared with NTG littermates or MLR14 TG mice. Histologic analysis of E15.5 MLR21 TG embryos disclosed a marked swelling in the differentiating fibers of the bow region and subcapsular fibers of the central zone, whereas the lens epithelium appeared morphologically normal. The lenticular changes, initiated early during lens development in TG MLR21 embryos, result in severe bilateral nuclear cataracts readily observable in neonates under normal rearing and dietary conditions. In contrast, TG MLR14 pups reared under standard conditions produced no lens opacity. CONCLUSIONS: Lens fiber swelling and related cataractous outgrowth positively correlated to the degree of lens bSMIT gene expression and intralenticular myo-inositol content. The affected (i.e., swollen) lens fibers appeared to be unable to cope with the water stress generated by the transgene-induced over-accumulation of myo-inositol and, as a result of this inability to osmoregulate, suffered osmotic damage due to water influx.

Animals↗
PubMed1998

Disregulation of ocular morphogenesis by lens-specific expression of FGF-3/int-2 in transgenic mice.

FGF-3, originally named int-2, was discovered as an oncogene frequently activated in mammary carcinomas resulting from the chromosomal integration of the mouse mammary tumor virus (MMTV). Int-2 was later designated FGF-3 based on sequence homology with other members of the fibroblast growth factor (FGF) family. FGF-1 is the prototypical member of the FGF family, and is the only family member which activates all known FGF receptor isoforms. Transgenic mice expressing in the lens a form of FGF-1 engineered to be secreted show premature differentiation of the entire lens epithelium. In contrast, transgenic mice engineered to secrete FGF-2 in the lens do not undergo premature differentiation of the lens epithelium (C. M. Stolen et al., 1997, Development 124, 4009-4017). To further assess the roles of FGFs and FGF receptors in lens development, the alpha A-crystallin promoter was used to target expression of FGF-3 to the developing lens of transgenic mice. The expression of FGF-3 in the lens rapidly induced epithelial cells throughout the lens to elongate and to express fiber cell-specific proteins including MIP and beta-crystallins. This premature differentiation of the lens epithelium was followed by the degeneration of the entire lens. Since FGF-1 and FGF-3 can both activate one FGF receptor isoform (FGFR2 IIIb) that is not activated by FGF-2, these results suggest that activation of FGFR2 IIIb is sufficient to induce fiber cell differentiation throughout the lens epithelium in vivo. Furthermore, transgenic lens cells expressing FGF-3 were able to induce the differentiation of neighboring nontransgenic lens epithelial cells in chimeric mice. Expression of FGF-3 in the lens also resulted in developmental alterations of the eyelids, cornea, and retina, and in the most severely affected transgenic lines, the postnatal appearance of intraocular glandular structures.

Animals↗
PubMed1996

Association of the type I regulatory subunit of cAMP-dependent protein kinase with cardiac myocyte sarcolemma.

Cardiac sarcolemmal vesicles purified from bovine and porcine left ventricles contained approximately 45 pmol of cAMP-dependent protein kinase (PKA) regulatory (R) subunit per milligram membrane protein based on [3H]cAMP-binding activity. Less than 26% of this activity was complexed with the catalytic subunit forming the type H holoenzyme of PKA. The remainder was contributed by the free type I R subunit (RI). Purification of sarcolemma with buffers containing 0.15 M NaCl instead of 0.75 M NaCl did not affect the ratio of RI to RII, nor did it increase the total amount of membrane-associated cAMP-binding or kinase activity. Canine, rabbit, and rat heart sarcolemma also contained RI, but in highly varying proportions compared with RII as determined by 8-N3-[32P]cAMP photoaffinity labeling. Analysis of sarcolemmal vesicles from isolated porcine ventricular myocytes demonstrated that this cell type was the source of the membrane-associated RI. The results indicate that sarcolemmal RI must be considered as a factor that could influence the varied responses of the heart to agents that elevate intracellular cAMP.

Adenosine Triphosphatases↗
PubMed1996

Differential expression of alpha A- and alpha B-crystallin during murine ocular development.

PURPOSE: To compare the temporal and spatial expression patterns of alpha A- and alpha B-crystallin mRNA during ocular development. METHODS: Tissue samples from embryonic day 9.5 (E9.5) through postnatal day 14 were collected from FVB/N strain mice. The specimens were fixed in paraformaldehyde, histologically processed, and assayed for alpha A- and alpha B-crystallin mRNA expression by in situ hybridization. RESULTS: During ocular development, alpha B-crystallin transcripts are present in the lens placode at E9.5. Transcripts of alpha A-crystallin are first observed in the lens cup at E10 to 10.5. During subsequent development of the lens, alpha A crystallin transcripts are most abundant in the fiber cells, and alpha B crystallin mRNA is preferentially expressed in epithelial cells. Transcripts of alpha A-crystallin were detected only in the lens. In contrast, alpha B-crystallin transcripts are present in retinal pigment epithelium, optic nerve, extraocular muscle, iris, ciliary body, cornea, and several nonocular sites, such as heart and nasal epithelium. CONCLUSIONS: Transcription of alpha B-crystallin precedes the expression of alpha A-crystallin during murine ocular development. Furthermore, the patterns of alpha A- and alpha B-crystallin expression in the lens are distinctive: alpha A is upregulated and alpha B is downregulated during prenatal fiber cell differentiation. These results indicate that the alpha-crystallin genes are not identically regulated either within or outside the lens.

Animals↗
PubMed1995

Expression of a truncated FGF receptor results in defective lens development in transgenic mice.

Members of the fibroblast growth factor (FGF) family are thought to initiate biological responses through the activation of cell surface receptors which must dimerize to transmit an intracellular signal. Mammalian lens epithelial cells respond to exogenous extracellular FGF, either in tissue culture or in transgenic mice, by initiating fiber cell differentiation. The role of FGF signalling in normal lens development was evaluated by lens-specific synthesis of a kinase-deficient FGF receptor type I (FGFR1) in transgenic mice. This truncated FGF receptor is thought to act as a dominant negative protein by heterodimerization with endogenous FGF receptors. The presence of transgenic mRNA in the lens was confirmed by in situ hybridization and by polymerase chain reaction amplification of reverse transcribed lens RNA (RT-PCR). The presence of transgenic protein was determined by Western blotting with antibodies to an extracellular domain of FGFR1. Three of four transgenic families expressing the truncated FGF receptor exhibited lens defects ranging from cataracts to severe microphthalmia. While the microphthalmic lenses displayed a normal pattern of differentiation-specific crystallin expression, the lens epithelial cells were reduced in number and the lens fiber cells displayed characteristics consistent with the induction of apoptosis. Our results support the view that FGF receptor signalling plays an essential role in normal lens biology.

Animals↗
PubMed1995

Extracellular FGF-1 acts as a lens differentiation factor in transgenic mice.

The vertebrate ocular lens undergoes a spatially defined pattern of differentiation which may be regulated by the ocular distribution of proteins from the fibroblast growth factor (FGF) family. The ability of altered FGF-1 (acidic FGF) distribution to disrupt the normal pattern of lens differentiation was evaluated by the production of transgenic mice which express FGF-1 under the control of the lens-specific alpha A-crystallin promoter. Since FGF-1 lacks a classical signal peptide consensus sequence, transgenic mice were also produced with a chimeric construct containing the signal peptide sequence of the FGF-4 gene fused in frame to the coding sequences of the FGF-1 cDNA in order to obtain extracellular expression of the transgene. The presence of transgenic mRNA and protein was confirmed by in situ hybridization, Western analysis and immunohistochemistry. The ocular histology of newborn and young adult transgenic mice expressing FGF-1 without a signal peptide appeared normal. In contrast, mice expressing secreted FGF-1 exhibited lens abnormalities including the elongation of anterior epithelial cells. Epithelial cell elongation was accompanied by expression of the fiber cell differentiation marker, beta-crystallin. These observations provide an in vivo demonstration that FGF-1 can induce anterior lens epithelial cells to express characteristics consistent with the onset of fiber cell differentiation. The transgenic induction of differentiation confirms that normal lens morphology reflects an asymmetric distribution of inductive factors within the eye.

Animals↗
PubMed1994

The value of duplex sonography after peripheral artery angioplasty in predicting subacute restenosis.

OBJECTIVE: The purpose of this study was to determine if abnormal findings on duplex sonographic examination after peripheral artery angioplasty correlate with the subsequent recurrence of a stenosis. SUBJECTS AND METHODS: We used duplex sonography to examine 35 stenoses in 23 patients within 48 hr after the patients had angioplasty to treat these stenoses. Patients were followed up for 3 years by using one or more of the following: assessment of signs and symptoms, monitoring of peripheral pulses, pulse volume recordings, and angiography. Life tables were constructed to compare long-term patency with the presence of abnormal findings seen on duplex sonograms. Abnormal findings at the dilated segment included a blood-flow velocity greater than 120 cm/sec or a residual elevated velocity ratio greater than 1.4 or 2.0 immediately after angioplasty. RESULTS: Twelve (34%) of 35 angioplasty sites showed recurrent stenosis before 36 months. Patency at 24 months was calculated for velocities less than 120 cm/sec vs velocities of 120 cm/sec or greater (41% vs 68%), for velocity ratios less than 1.4 vs ratios of 1.4 or greater (63% vs 57%), and for velocity ratios less than 2.0 vs ratios of 2.0 or greater (54% vs 74%). We found no significant difference in patency between those patients with normal findings and those with abnormal findings on duplex sonographic examination after angioplasty. CONCLUSION: Abnormal findings on duplex sonograms obtained immediately after peripheral angioplasty cannot be used to predict subacute restenosis.

Angioplasty, Balloon↗
PubMed1993

[125I]Vasoactive intestinal peptide binding in rodent suprachiasmatic nucleus: developmental and circadian studies.

The suprachiasmatic nucleus (SCN) of rat and hamster have been studied extensively and shown to play critical roles in circadian rhythmicity. [125I]Vasoactive intestinal peptide (VIP) binding levels are high in the rat SCN, suggesting that VIP receptors may be an important component of SCN function. In contrast to previously demonstrated diurnal variations in VIP immunoreactivity and VIP mRNA, the present study found [125I]VIP binding to be stable across the light-dark cycle in both rat and hamster SCN. High [125I]VIP labeling appeared to be coextensive with the rat SCN but extended somewhat beyond the cytoarchitectonic boundaries of the hamster SCN. Binding density in hamster SCN was slightly higher than in rat. In the developing rat SCN, [125I]VIP binding levels distinguished the SCN on embryonic day 18, and appeared to increase to postnatal day 10 before declining to adult levels. The early presence of [125I]VIP binding suggests possible involvement of VIP receptors in fetal entrainment of circadian rhythms.

Animals↗
PubMed1993

Genetic control of ocular morphogenesis: defective lens development associated with ocular anomalies in C57BL/6 mice.

Congenital ocular defects, including microphthalmia, anophthalmia and cataracts appear in a small proportion (5-15%) of the C57BL/6 inbred strain of laboratory mice. Previous work from this laboratory utilizing C57BL/6 in equilibrium with DBA/2 allophenic (mosaic) mice suggested that the underlying cause of these defects may be a deficiency in C57BL/6 lens development. In these C57BL/6 in equilibrium with DBA/2 allophenic animals, the major proportion of the lens cells were of the DBA/2 strain, in contrast to the other ocular and non-ocular tissues examined where no strain-specific competition was evident. Results presented here reinforce the notion that C57BL/6 lens cells are developmentally retarded. In allophenic combination with another strain, A/J, cells of the C57BL/6 strain were, again, markedly underrepresented in the mosaic lens, whereas no such genotypic specificity was evident in any of the other ocular and non-ocular tissues. Thus, the previous observation of a C57BL/6 deficiency in mosaic lens is more likely a consequence of low C57BL/6 competitiveness rather than some peculiar hyperactivity of the cells of the partner strains. Histological examination of C57BL/6, DBA/2 and A/J pure strain embryos indicated marked differences in the lens development of C57BL/6 embryos compared to the other two strains. For the first 3 days after lens invagination, the cross-sectional areas of DBA/2 lenses were two- to three-fold larger than those of C57BL/6 lenses at comparable stages of development, whereas no comparable difference was noted in the optic cup development in these two strains. This leads to a much higher lens/optic cup size ratio in DBA/2 embryos than C57BL/6 embryos. Scanning electron microscopy also revealed that lens invagination is much less extensive in the C57BL/6 embryos than the A/J embryos. The smaller C57BL/6 lens vesicle also remains deeper in the optic cup than those of the DBA/2 or A/J embryos of comparable developmental age. This is likely to result in the occasional complete envelopment of the entire lens vesicle by the optic cup and lead to arrested ocular development.

Animals↗
PubMed1993

LC studies on the potential interaction of paraben preservatives with sorbitol and glycerol.

Pharmaceutical formulations often contain one or more paraben preservatives in conjunction with a polyol such as sorbitol or glycerol. In one of these experimental formulations a number of unknown polar peaks have been detected near the solvent front by reversed-phase LC. These degradation products were not attributable to the active drug component or a hydrolysis product. The possibility of an interaction between the polyols and paraben preservatives has been explored using a three-variable, two-level, factorial design to determine the relative significance of the factors involved in the formation of these unknown peaks. The factors examined were pH, temperature and the ratio of polyol to paraben. This study has shown that pH and temperature are key factors affecting the formation of these unknown peaks. On the basis of these results suitable conditions can be suggested for minimizing the production of these unknown peaks. It seems clear that a number of pharmaceutical formulations containing a polyol and a paraben would present potential problems for assay validation on storage owing to the formation of these degradation products, particularly if the drug component is polar and elutes near the solvent front.

Chromatography, Liquid↗
PubMed1992

Duplex sonography of the carotid arteries.

Duplex sonography is the best noninvasive modality for investigation of possible carotid artery stenosis. By using the above described techniques, almost all significant stenoses can be detected and categorized correctly. Knowledge of common pitfalls in the performance and interpretation of the examination is essential to avoid misdiagnosis. Color imaging is a helpful addition to conventional duplex imaging, but is not essential to the performance of high-quality examinations.

Carotid Arteries↗
PubMed1992

Peripheral arterial Doppler ultrasonography: diagnostic criteria.

Fifty-one patients (86 legs) were studied from the aorta to the popliteal artery with both duplex ultrasonography and arteriography with the intention of establishing diagnostic ultrasonographic criteria for degrees of stenosis greater than 50% and determining the effect of multi-segment disease on the accuracy of these criteria. Receiver operator characteristic curves were used to determine diagnostic criteria for greater than 50%, greater than 70%, greater than 90% diameter stenoses or occlusions. A stenosis of greater than 70% was diagnosed either if the peak systolic velocity was more than 160 cm/sec (sensitivity 77%, specificity 90%) of if there was an increase in peak systolic velocity of 100% with respect to the arterial segment above the stenosis (sensitivity 80%, specificity 93%). Sequential stenoses were detected with lesser sensitivities. Ratio criteria were more accurate than peak systolic velocity for all degrees of stenosis. For detection of occlusion, duplex ultrasonography was 92% sensitive and 99% specific.

Arterial Occlusive Diseases↗
PubMed1992

Acute limb ischemia.

Acute ischemia of the extremity may be due to arterial occlusion from spontaneous thrombosis, embolus, arterial bypass graft thrombosis, trauma, or spasm. The presence of occlusion or stenosis can be determined noninvasively with the use of duplex Doppler ultrasonography. Most patients will require arteriography prior to thrombolytic or surgical therapy. New techniques, such as percutaneous aspiration thrombolectomy, expand the role of radiologic percutaneous therapy of the acutely ischemic limb. Prompt diagnosis and therapy are required to avoid limb loss or systemic metabolic complications from reperfusion of a dying limb.

Angioplasty, Balloon↗
PubMed1990

Evaluation of the peripheral arteries with duplex US after angioplasty.

Duplex ultrasound (US) was performed in 22 patients before and immediately after percutaneous transluminal balloon angioplasty (PTA) for peripheral artery stenoses or occlusions. Two patients underwent PTA on two occasions, and one patient underwent PTA of each lower extremity, resulting in 25 duplex US imaging pairs at a total of 40 PTA sites. The criterion for significant stenosis on duplex US studies was an increase in peak systolic velocity of at least 100% compared with the velocity in the arterial segment proximal to the lesion (velocity ratio, greater than or equal to 2.0). For 22 paired duplex US studies, pulse volume recordings or ankle-brachial indexes were obtained before and after PTA. After PTA, eight patients had residual high velocity ratios at 12 PTA sites. Only two of these patients had a residual hemodynamic stenosis as determined with post-PTA arteriography, pulse volume recordings, or ankle-brachial indexes. In this study, duplex US that was performed immediately after PTA for evaluation of residual stenosis was frequently misleading.

Angiography↗