Emersion behaviour underlies variation in gill morphology and aquatic respiratory function in the amphibious fish Kryptolebias marmoratus.

Fishes acclimated to hypoxic environments often increase gill surface area to improve O2 uptake. In some species, surface area is increased via reduction of an interlamellar cell mass (ILCM) that fills water channels between gill lamellae. Amphibious fishes, however, may not increase gill surface area in hypoxic water because these species can, instead, leave water and breathe air. To differentiate between these possibilities, we compared wild amphibious mangrove rivulus Kryptolebias marmoratus from two habitats that varied in O2 availability - a hypoxic freshwater pool versus nearly anoxic crab burrows. Fish captured from crab burrows had less gill surface area (as ILCMs were enlarged by ∼32%), increased rates of normoxic O2 consumption and increased critical O2 tension compared with fish from the freshwater pool. Thus, wild mangrove rivulus do not respond to near-anoxic water by decreasing metabolism or increasing O2 extraction. Instead, fish from the crab burrow habitat spent three times longer out of water, which probably caused the observed changes in gill morphology and respiratory phenotype. We also tested whether critical O2 tension is influenced by genetic heterozygosity, as K. marmoratus is one of only two hermaphroditic vertebrate species that can produce both self-fertilized (inbred) or out-crossed (more heterozygous) offspring. We found no evidence for inbreeding depression, suggesting that self-fertilization does not impair respiratory function. Overall, our results demonstrate that amphibious fishes that inhabit hypoxic aquatic habitats can use a fundamentally different strategy from that used by fully aquatic water-breathing fishes, relying on escape behaviour rather than metabolic depression or increased O2 extraction ability.

Angiotensin II induces delayed mitogenesis and cellular proliferation in rat aortic smooth muscle cells. Correlation with the expression of specific endogenous growth factors and reversal by suramin.

By means of a rat aortic smooth muscle (RASM) cell culture model, the effects of angiotensin II (AII) on early proto-oncogene gene expression, DNA synthesis, and cell proliferation were measured and compared to known mitogens. In 24-h [3H]-thymidine incorporation assays, AII was found to be a weak mitogen when compared to potent mitogens such as fetal bovine serum and platelet-derived growth factor (PDGF). In contrast, when assays were carried out for 48 h, AII induced a significant dose-dependent stimulation of DNA synthesis, which more than doubled at 3 nM AII, and was maximal (five- to eightfold above control) at 100 nM AII. Treatment of cells with the AII type 1 receptor antagonist losartan inhibited the mitogenic effects of AII. AII also stimulated smooth muscle cell proliferation, as indicated by an absolute increase in cell number after AII stimulation of RASM cells for 5 d. AII stimulation of RASM cell growth correlated with the increased expression of specific endogenous growth factors, including transforming growth factor beta 1 (TGF-beta 1) and PDGF A-chain. However, addition of either PDGF- or TGF-beta 1-neutralizing antibodies failed to significantly reduce the delayed mitogenic effects induced by AII. In contrast, we found that AII-stimulated mitogenesis could be inhibited in a dose-dependent manner by the growth factor inhibitor drug suramin. Taken together, our results indicate that enhanced endogenous growth factor expression may represent the direct mechanism by which AII promotes smooth muscle cell growth in some vascular hyperproliferative diseases.

Stimulation of activin A expression in rat aortic smooth muscle cells by thrombin and angiotensin II correlates with neointimal formation in vivo.

Vasoactive GTP-binding protein-coupled receptor agonists (e.g., angiotensin II [AII] and alpha-thrombin) stimulate the production of mitogenic factors from vascular smooth muscle cells. In experiments to identify mitogens secreted from AII- or alpha-thrombin-stimulated rat aortic smooth muscle (RASM) cells, neutralizing antibodies directed against several growth factors (e.g., PDGF and basic fibroblast growth factor [basic FGF]) failed to inhibit the mitogenic activity of conditioned media samples derived from the cells. In this report, we found that polyclonal neutralizing antibodies directed against purified human placental basic FGF reduced the mitogenic activity of AII-stimulated RASM cell-conditioned media and in immunoblot experiments identified a 26-kD protein (14 kD under reducing conditions) that was distinct from basic FGF. After purification from RASM cell-conditioned medium, amino acid sequence analysis identified the protein as activin A, a member of the TGF-beta superfamily. Increased activin A expression was observed after treatment of the RASM cells with AII, alpha-thrombin, and the protein kinase C agonist PMA. In contrast, PDGF-BB or serum caused only a minor induction of this protein. Although activin A alone only weakly stimulated RASM cell DNA synthesis, it demonstrated a potent comitogenic effect in combination with either EGF or heparin-binding EGF-like growth factor in the RASM cells, increasing DNA synthesis by up to fourfold. Furthermore, in a rat carotid injury model, activin A mRNA was upregulated within 6 h after injury followed by increases in immunoreactive protein detected in the expanding neointima 7 and 14 d later. Taken together, these results indicate that activin A is a vascular smooth muscle cell-derived factor induced by vasoactive agonists that may, either alone or in combination with other vascular derived growth factors, have a role in neointimal formation after arterial injury.

Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist.

Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately 15-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic fibroblast growth factor (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombin was delayed by an additional 3-6 h. Delayed mitogenesis correlated with the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle cells, as assayed using Swiss 3T3 fibroblasts. This activity was not inhibited by neutralizing antibodies directed against PDGF or bFGF. Furthermore, in the Swiss 3T3 cells, simple addition of either alpha-thrombin or SFLLRNP failed to elicit a significant mitogenic response. In signal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 75, 120, and 190 kD, respectively, as assessed by antiphosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB addition. Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44mapk and p42mapk was also observed. However, the time course and duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB. Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferation is delayed and requires the de novo expression of one or more autocrine mitogens. In addition, the rapid induction of discrete intracellular signaling mechanisms by thrombin, including the Raf-1/MAP kinase pathway, appears to be insufficient alone to promote vascular smooth muscle cell mitogenesis.

Diagnosis from fundus photographs.

AIM: To investigate the way in which ophthalmologists observe fundi and make diagnoses from their observations. METHODS: A set of 12 test photographs was presented to 9 ophthalmologists. The subjects were asked to identify the features in the photographs that are important for forming a diagnosis and were also asked to form differential diagnoses. The scanpaths of the subjects were recorded during their inspection of the photographs. Subsequently, they were asked to trace over the important features of four of the photographs. RESULTS: The correctness of the diagnoses was described by weighted numerical scores. Differential diagnoses made after 30 s of inspection were significantly better than those made after 5 s. Irrespective of correctness, the reported diagnoses were dominated by the most obvious features of the photograph. Incorrect diagnoses were made either because the subjects failed to identify the significant features of the photograph or because they failed to comprehend the significance of the identified features. CONCLUSION: Accurate funduscopy involves both perception of diagnostic features and cognitive interpretation of these features. Verbal reports, eye movement recordings and tracings reveal the features and interpretations used to make a diagnosis. These techniques will be used in a subsequent study to evaluate the relative contributions of formal training and experience to the development of diagnostic skills.

The achiasmia spectrum: congenitally reduced chiasmal decussation.

AIM: To describe the clinical spectrum of achiasmia, a congenital disorder of reduced relative decussation at the optic chiasm. METHODS: A retrospective case note and patient review of nine children (four boys). Achiasmia was defined by the combination of a characteristic asymmetry of the monocular visual evoked potential (VEP) response to flash and neuroimaging showing reduced chiasmal size. RESULTS: Three of the children had an associated skull base encephalocele with agenesis of the corpus callosum. In two patients achiasmia was associated with septo-optic dysplasia. Three patients had no neuroimaging abnormalities other than reduced chiasmal size and have no known pituitary dysfunction. One child had multiple physical deformities but the only brain imaging abnormality was reduced chiasmal size. CONCLUSIONS: Some children with disorders of midline central nervous system development, including septo-optic dysplasia and skull base encephaloceles, have congenitally reduced chiasmal decussation. Reduced relative decussation may co-exist with overall chiasmal hypoplasia. Children with an apparently isolated chiasmal decussation deficit may have other subtle neurological findings, but our clinical impression is that most of these children function well.

Endothelin-1 and angiotensin-II stimulate delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for common signaling mechanisms.

The vasoactive peptides endothelin-1 (ET-1) and angiotensin-II (AII) have been implicated in chronic hypertension and may play important roles in related vascular diseases such as restenosis and atherosclerosis. Using a rat aortic smooth muscle (RASM) cell model, both ET-1 and AII induced concentration-dependent delayed increases in DNA synthesis relative to that in the serum-deprived controls. Stimulation of DNA synthesis was maximal at 100 nM for each peptide. All treatment of RASM cells resulted in a greater mitogenic effect (4- to 7-fold) than that observed for ET-1 (3-fold). When added in the presence of AII, ET-1 had a supplemental effect on DNA synthesis (5- to 10-fold above control). Although RASM cells expressed both ETA and AT1 receptors, radioligand binding experiments indicated that approximately 10-fold as many AT1 receptors as ETA receptors were present. In signal transduction studies, ET-1 and AII each elicited concentration-dependent increases in the intracellular Ca2+ concentration. ET-1 and AII also stimulated phosphoinositide metabolism and phosphorylation of a specific substrate for protein kinase-C. The release of total inositol phosphates in response to ET-1 and AII was concentration dependent and inhibited by the ETA receptor-selective antagonist BQ-123 and the AT1 receptor-selective antagonist losartan, respectively. In addition, tyrosine phosphorylation of 120- and 75-kilodalton proteins as well as the mitogen-activated protein kinases p44mapk and p42mapk was observed within 5 min of the addition of either ET-1 or AII. Taken together, these data indicate that ET-1 and AII may promote smooth muscle cell growth through common intracellular signaling mechanisms.

Cyclosporin A directly inhibits human B-cell proliferation by more than a single mechanism.

Cyclosporin A (CsA) is a potent immunosuppressive agent that inhibits T-cell proliferation and lymphokine production. There is less information on the direct effect of CsA on B-cells. We investigated the proliferative responses of human tonsillar B-lymphocytes to a "T dependent" mitogen, pokeweed mitogen (PWM), and to a "T independent" mitogen, Staphylococcus aureus (SA). Both responses were strongly inhibited by CsA. Nonspecific cytotoxicity was ruled out, and the inhibition was not reversed by adding IL1, IL2, or BCGF individually or in combination. Maximal inhibition of the PWM response occurred when CsA was added early in the culture period. Cyclosporin A added 18 hours after the start of culture was less effective, and adding CsA after 36 hours resulted in only minimal inhibition. However, with SA as mitogen, addition after 36 hours still affected substantial inhibition. These results, on the time of action and resistance to reversal by exogenous growth factors, suggest that CsA can directly inhibit human B-cells by a mechanism similar to its action on T-lymphocytes, blocking an early event critical to entry into cell cycle, but an additional mechanism of inhibition later in the cell cycle may also operate when the proliferative signal is provided by the T-independent mitogen SA.

Polyunsaturated fatty acids in the food chain in the United States.

In the United States, intake of n-3 fatty acids is approximately 1.6 g/d ( approximately 0.7% of energy), of which 1.4 g is alpha-linolenic acid (ALA; 18:3) and 0.1-0.2 g is eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6). The primary sources of ALA are vegetable oils, principally soybean and canola. The predominant sources of EPA and DHA are fish and fish oils. Intake data indicate that the ratio of n-6 to n-3 fatty acids is approximately 9.8:1. Food disappearance data between 1985 and 1994 indicate that the ratio of n-6 to n-3 fatty acids has decreased from 12.4:1 to 10.6:1. This reflects a change in the profile of vegetable oils consumed and, in particular, an approximate 5.5-fold increase in canola oil use. The ratio of n-6 to n-3 fatty acids is still much higher than that recommended (ie, 2.3:1). Lower ratios increase endogenous conversion of ALA to EPA and DHA. Attaining the proposed recommended combined EPA and DHA intake of 0.65 g/d will require an approximately 4-fold increase in fish consumption in the United States. Alternative strategies, such as food enrichment and the use of biotechnology to manipulate the EPA and DHA as well as ALA contents of the food supply, will become increasingly important in increasing n-3 fatty acid intake in the US population.

Binding of tissue plasminogen activator to cultured human fibroblasts.

The binding of 125I-labeled, one-chain tissue plasminogen activator (t-PA) by WI-38 cultured human lung fibroblasts was investigated. Binding of t-PA to WI-38 monolayers was specific, saturable and temperature dependent. One and two-chain t-PAs were comparable in their ability to compete with 125I-labeled, one-chain t-PA for binding to fibroblasts, while no inhibition of binding was observed with a 500-fold molar excess of urokinase. Studies with various compounds suggest that neither the catalytic site, the fibrin binding site, nor the carbohydrate moieties on t-PA are involved in its binding to WI-38 cells. At higher temperatures, the amount of cell-bound 125I-t-PA that was removed by either incubation in binding buffer containing an excess of unlabeled t-PA, or by brief treatment with acidic buffer, was small (approximately 20%) suggesting that much of the t-PA is internalized. Electrophoretic analysis of extracts prepared from cells that had been incubated with 125I-t-PA revealed the presence of a major band of 70,000 Mr, which corresponds to intact t-PA. Our results suggest that WI-38 fibroblasts are capable of binding and internalizing t-PA, and that these processes involve a receptor site specific for t-PA.

Novel cardiovascular actions of the activins.

Proliferation and directed migration of vascular cells are key components in vascular diseases such as atherosclerosis and restenosis following percutaneous transluminal coronary angioplasty. However, the precise cellular and molecular mechanisms involved in the control of vascular cell proliferation or migration at the tissue level remain largely undefined. Molecules contributing to these processes are elaborated by distinct cell types and act in both autocrine and paracrine modes. They include two broad classes, polypeptide growth factors and vasoactive G-protein-coupled receptor (GPCR) agonists. Examples of the former, such as platelet-derived growth factor, bind to and activate cell surface receptor tyrosine kinases, initiating intracellular biochemical signaling pathways associated with cell proliferation or migration. In contrast, recent evidence suggests that vasoactive GPCR agonists (e.g. angiotensin II, endothelin-1, alpha-thrombin) elicit cell growth indirectly by inducing the production of autocrine or paracrine factors in vascular cells. Recent studies have identified activin A as a novel component of conditioned medium obtained from GPCR agonist-stimulated vascular smooth muscle cells (SMCs). Although activin A alone only weakly stimulated rat aortic SMC DNA synthesis, it demonstrated a potent co-mitogenic effect in combination with either epidermal growth factor (EGF) or heparin binding EGF-like growth factor in these cells, increasing DNA synthesis by up to 5- and 4-fold respectively. Furthermore, in a rat carotid-injury model, activin A mRNA was upregulated within 6 h after injury, followed by increases in immunoreactive protein detected in the expanding neointima 7 to 14 days later. Taken together, these results indicate that activin A is a common vascular SMC-derived growth factor induced by vasoactive agonists that may, either alone or in combination with other factors, contribute to fibroproliferative vascular diseases.

Effects of thrombin receptor activating peptide on phosphoinositide hydrolysis and protein kinase C activation in cultured rat aortic smooth muscle cells: evidence for "tethered-ligand" activation of smooth muscle cell thrombin receptors.

Phosphoinositide hydrolysis and protein kinase C (PKC) activation were examined in response to treatment of rat aortic smooth muscle cells with alpha-thrombin and a seven amino acid thrombin receptor activating peptide (TRAP-7; SFLLRNP). alpha-Thrombin and TRAP-7 stimulated total inositol phosphate (IP) accumulation and phosphorylation of a specific endogenous substrate for activated PKC. Acetylated TRAP-7 and "reverse" TRAP (FSLLRNPNDKYEPF) were ineffective in stimulating signal transduction. The active site inhibitor, MD805 (argatroban), and the anion-binding exosite inhibitor, BMS 180,742, reduced the IP response to alpha-thrombin in a concentration-dependent manner. In contrast, the TRAP-7-induced IP response was not affected by either inhibitor. These data are consistent with the tethered-ligand hypothesis for thrombin receptor activation in rat aortic smooth muscle cells.

The infant corneal endothelium.

Specular microscopy of the in vivo corneal endothelium of 48 clinically normal eyes of 31 infants less than 1 year old revealed a regular mosaic of small cells. The cell population density of individuals varied greatly, as it does in age-related adults. Reexamination of five eyes indicated a reduction of the cell population density during the first year. This change could be accounted for by corneal growth in the absence of endothelial mitoses and not necessarily by true cell loss. There were morphologic indications of mitoses, but their interpretation is open to doubt.

Oral contraceptive use by teenage women does not affect peak bone mass: a longitudinal study.

OBJECTIVE: To determine the effect of oral contraceptive pill (OCP) use during adolescence on peak bone mass. DESIGN: Longitudinal observational study. SETTING: Academic clinical research center. PATIENT(S): Sixty-two non-Hispanic, white females in The Penn State Young Women's Health Study, who were studied for 8 years during ages 12-20. INTERVENTION(S): There were 28 OCP users, who used OCPs for a minimum of 6 months and were still using at age 20, and 34 nonusers who had never used OCPs. MAIN OUTCOME MEASURE(S): Total body bone, dedicated hip bone, and body composition measurements were made by dual-energy roentgenogram absorptiometry. RESULT(S): The OCP users and nonusers did not differ at entry in anthropometric, body composition, or total body bone measurements. By age 20, the average duration of OCP use by the user group was 22 months. At age 20, the groups remained indistinguishable in anthropometric, body composition, total body, and hip bone measures, and in age of menarche and sports exercise scores. CONCLUSION(S): Oral contraceptive pill use by healthy, white, teenage females does not affect acquisition of peak bone mass.

PIP: This longitudinal observational study determined the effect of oral contraceptive (OC) use during adolescence on peak bone mass (PBM). The sample comprised 62 non-Hispanic, White females in The Penn State Young Women's Health Study, who were studied for 8 years between the ages of 12 and 20. There were 28 OC users who used OCs for a minimum of 6 months and were still using them at age 20, and 34 nonusers who had never used the regimen. Total body bone, dedicated hipbone, and body composition measurements were made by dual-energy roentgenogram absorptiometry. There was no difference between OC users and nonusers in the anthropometric, body composition, or total body bone measurements. By age 20, the average duration of OC use by the user group was 22 months. At this age, the groups remained indistinguishable in anthropometric, body composition, total body, and hipbone measurements, and in age of menarche and sports exercise scores. These findings suggest that OC use by healthy, White, teenage females does not affect acquisition of PBM.

A syndrome of congenital retinal dystrophy and saccade palsy--a subset of Leber's amaurosis.

Three children who presented in infancy with a severe visual defect and absent or barely recordable electroretinograms, with relatively well preserved visually evoked cortical potentials, were subsequently found to have vertical and horizontal saccade palsies with head thrusts but relatively good visual acuity. These children, who were clearly different from other infants with congenital retinal dystrophy, were also developmentally delayed and had systemic motor and speech defects, but their visual prognosis was relatively good. The recognition of their saccade palsy was delayed because their poor visual attention in infancy was ascribed purely to the tapetoretinal degeneration. We consider these patients represent a clear subset of those patients who are diagnosed as having congenital retinal dystrophy or Leber's amaurosis.

Histiocytosis X: an ophthalmological review.

Of 76 children with histiocytosis X 18 had orbital involvement, and four developed additional neuro-ophthalmic complications. No instance of intraocular involvement was detected. Among those patients with ophthalmic involvement the main problems were bilateral or unilateral proptosis, ptosis, papilloedema, optic atrophy, and seventh nerve palsy. Only one patient developed a severe visual defect. Management of the ophthalmological complications depends not only on the extent of the orbital disease but also on the degree of systemic involvement. Overall management by a paediatric oncologist is mandatory.

Localising patterns of optic nerve hypoplasia--retina to occipital lobe.

Six cases are presented which provide clinical evidence that optic nerve hypoplasia can occur as a result of a lesion at any site in the developing visual system. The mechanisms of hypoplasia are discussed in the light of recent understanding of optic nerve development.

Bilateral macular dysplasia ('colobomata') and congenital retinal dystrophy.

Three unrelated patients with bilateral macular dysplasia ('colobomata') with no relevant family history were found to have absent or substantially abnormal electroretinograms, implying that there was an associated retinal dystrophy. This may suggest that the macular lesions are associated with a global failure of retinal development, with a regional preponderance rather than a purely localised cause such as an intrauterine infection. It is important to distinguish between congenital infections such as toxoplasmosis and developmental macular colobomata, which have a somewhat similar ophthalmoscopic appearance as a cause of bilateral macular abnormalities seen in young children, since they have different implications for genetic advice and future ophthalmic care.

Albinism in childhood: a flash VEP and ERG study.

Flash visual evoked potentials (F. VEPs) and electroretinograms (ERGs) were recorded in a total of 20 young children with albinism (age range 5 months to 11 years, mean 4 years). All recordings were made without sedation. There were 13 oculocutaneous cases (one with Hermansky-Pudlak syndrome) and seven ocular albinos. Monocular flash stimulation commonly elicited an asymmetrical occipital VEP distribution with a well lateralised component at around 80 ms which was of opposite polarity in a comparison of VEPs from each eye. None of the normally pigmented matched controls or obligate female carriers showed this anomalous distribution. The albino electroretinogram, compared with controls, recorded under fully darkened conditions had a significantly larger a wave and significantly shorter latencies for both a and b waves. The accentuated ERG and asymmetrical VEP recorded in infants and young children with albinism permits distinction of these patients from those with congenital cone dysfunction and idiopathic nystagmus, with whom they may be confused by a clinical examination only.

Spontaneous reversal of nystagmus in the dark.

AIM: To report five children with horizontal jerk nystagmus in whom eye movement recordings in the dark revealed a spontaneous reversal in the direction of the nystagmus beat. Three patients were blind in one eye and were diagnosed as having a manifest latent nystagmus (MLN), and two patients had strabismus and congenital nystagmus (CN). METHODS: Eye movements were recorded using DC electro-oculography with simultaneous video recording, including infrared recording in total darkness. RESULTS: Four patients had decelerating velocity slow phase jerk nystagmus when recorded under natural lighting conditions; the fifth case had accelerating velocity and linear slow phase jerk nystagmus. Under absolute darkness, nystagmus reversed in direction of beat with a mixture of linear and decelerating velocity slow phase waveforms. One child with unilateral anophthalmos could wilfully reverse the beat direction of his nystagmus by trying to look with his blind eye in the light and dark. CONCLUSIONS: These observations support the theory that LN/MLN beat direction is determined by the "presumed" viewing eye and may be consciously controlled. The spontaneous reversal of beat direction in the dark suggests eye dominance is predetermined. Eye movement recordings identified mixed nystagmus waveforms indicating that CN (accelerating velocity slow phases) and LN/MLN (linear/decelerating velocity slow phases) coexist in these subjects.