Flow measurement using duplex Doppler ultrasound: haemodynamic changes in patients with colorectal liver metastases.

Duplex Doppler ultrasound has been used to make measurements of liver blood flow. The relationship between time average velocity as measured using pulsed Doppler and the true mean velocity was determined using a flow phantom. Correction factors were applied to measured time average velocities of blood in the hepatic artery and portal vein. Blood flow was calculated from the product of corrected velocities and the cross-sectional area of the vessels. There was no significant difference in total liver blood flow for patients with colorectal liver metastases when compared with controls. However, the ratio of the hepatic arterial flow to the sum of the portal and hepatic arterial flows (Doppler perfusion index, DPI) was markedly elevated (P < 0.0001) in the patients with metastases when compared with the controls. The DPI values for a third group of subjects who had undergone resection for colonic cancer but who had no proven liver metastases overlapped the values for the other two groups. When attention is paid to technique, the procedure can detect and quantify the changes in liver blood flow which occur in the presence of metastases.


A thermal clearance probe for continuous monitoring of cerebral blood flow.

Thermal clearance has been used as a measure of tissue blood flow for over 60 years. We have developed a probe which can be used to monitor cerebral cortical thermal clearance in patients suffering from acute neurosurgical disorders. Despite its limitations of size, invasiveness, small sample volume, and difficult quantification it has proved a reliable method, showing a good correlation between measured thermal clearance and the onset of clinical signs of ischaemia.


Antimony electrode for the determination of the intramyocardial pH during open-heart surgery.

The development of an antiomony electrode (Sb) and a pH meter as a substitute for a glass electrode(G) to measure the intramyocardial pH is reported, and the results of their clinical application. The determination of the pH of CPD (citrate-phosphate-dextrose) blood by Sb showed small differences when compared to G (less than 0.1). Sb was also temperature sensitive. The temperature coefficient of Sb was determined in buffers by varying their temperatures, and an antimony-thermocouple electrode and a temperature-compensated pH meter were subsequently constructed. In CPD blood of varying temperatures Sb showed similar tendencies to G. The results of the application of the new apparatus to 28 patients were statistically no different from those obtained previously by G on 22 patients, either in baseline pH or its fall following aortic cross-clamping. It is concluded that although Sb is not as accurate as G, it is a reasonable alternative for the determination of myocardial acidosis during open-heart surgery.


Spectrophotometric measurements of haemoglobin saturation and concentration in skin during the tuberculin reaction in normal human subjects.

A non-invasive technique employing light-guide spectrophotometry is described for the measurement of haemoglobin concentration and oxygenation in human skin. Measurements were carried out in the visible wavelength range (500-620 nm) and a series of experiments were carried out in vitro and in vivo in order to calibrate the system. Indices were derived for the measurement of relative haemoglobin concentration and absolute oxygen saturation. The technique was applied to measure the changes in these parameters occurring during the course of the tuberculin reaction in human skin. The results are compared with those from laser Doppler flowmetry and transcutaneous oxygen measurements which were carried out concurrently. Divergence between the intracapillary and tissue oxygenation during the course of the reaction provides evidence for the existence of increased diffusion resistance for oxygen; a model is proposed. The study demonstrates the potential clinical usefulness of light-guide spectrophotometry for the non-invasive investigation of tissue oxygen supply.


Effects of myocardial electrotonic interaction on the sequence of excitation and repolarisation and on T wave polarity. Computer modelling experiments.

A computer model of a 3-dimensional rectangular block of myocardial tissue (3969 cells) has been used to investigate the influence on excitation and repolarisation sequences and on the modelled electrocardiographic T wave of (a) electrotonic interaction, (b) intrinsic distribution of refractoriness, and (c) the speed of repolarisation of action potentials. The model allowed electrotonic interactions to be investigated separately during the depolarisation and repolarisation phases. Scales of 14 values of the strength of electrotonic interaction during the depolarisation phase, 14 values of the strength of electrotonic interaction during the repolarisation phase, 3 shapes of action potential, and 5 distributions of tissue refractoriness were selected and all 2940 combinations were examined. In each experiment, the tissue model was artificially excited and the resulting excitation and repolarisation sequences were simulated. The results of the study suggested that electrotonic interactions between excited cells can cause non-uniform speed of propagation which, by means of the phase shifts of action potentials, contributes to the inversion of the repolarisation sequence and to the physiologic orientation of T waves. Experiments with this model did not support the hypothesis that simple electrotonic smoothing of the differences in repolarisation phases due to the excitation phase shift of action potentials reverses the repolarisation sequence and explains T wave polarity.


An error study on some linear reconstruction algorithms for electrical impedance tomography.

An error analysis on the Poisson-type linear reconstruction algorithms is performed. Three types of error are discussed and it is found that re-scaling error is dominant in most cases. It is also found that the commonly used small perturbation assumption in obtaining the Poisson's equation often fails but, due to the dominance of the re-scaling error, the reconstruction of the relative impedance distribution is still feasible by using the linear algorithms. The error analysis leads to a useful understanding of the mechanism of linear reconstruction approaches.


Changes of erythropoietic and storage iron components in certain clinical situations as evaluated by ferrokinetic investigations.

A method is described for the quantitative analysis of in vivo organ measurements of 59Fe activity in ferrokinetic investigations. The time-activity curves obtained by sequential surface monitoring over sacrum, liver and spleen can be resolved quantitatively into their different components contributing to the recorded count rate. The analysis is performed in three steps: (1) Count rate contributions from activity in other organs and from scattered radiation from regions outside the organ under investigation are corrected. (2) Distinction is made between radioactivity uptake of the tissue and radioactivity contained in the perfusing blood. (3) The resulting net organ activity is then further resolved into an erythropoietic and a storage iron component by use of a computer program (SAAM-27) and assuming a compartmental model for internal iron exchange. The method was tested in three groups of patients with different haematological disorders and in normal controls. Characteristic patterns of the parameters are found for different diseases and the results correlated well with the clinical findings. It is concluded that in vivo organ measurements of 59Fe activity, when performed and analysed with sufficient care, can provide an insight into the dynamics of the iron exchange that is taking place in an organ. This analytical approach may improve the diagnostic predictions of ferrokinetic investigations.


Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells.

Endothelin-like immunoreactivity was detected in human (15.6 +/- 2.7 pg/ml) and bovine (11.1 +/- 0.98 pg/ml) aqueous humour of the eye. These concentrations are 2-3 times higher than the corresponding plasma levels. Cultured human nonpigmented ciliary epithelial cells released endothelin-like immunoreactivity with a maximum of 2.1 +/- 0.32 pg/(cm2* 48 h). The release was stimulated by fetal calf serum, thrombin, carbachol and phorbol ester and blocked by cycloheximide. Immunocytochemistry showed cytoplasmic staining of cultured human nonpigmented ciliary epithelial cells for endothelin-1. Endothelin-1 was shown to induce contractions in isolated human ciliary muscle by isometric force measurements. Endothelin in the aqueous humour may play a role in the regulation of intraocular pressure.


Intravitreal and subretinal proliferation induced by platelet-rich plasma injection in rabbits.

We developed an experimental model of proliferative vitreoretinopathy (PVR) in albino rabbits by combining some factors suspected of causing the disease. Sixty nine eyes divided into six groups served as controls (Groups C 1-6). Forty nine eyes were divided into four experimental groups (Groups E 1-4). Group E1 (n = 12) was injected with 0.15 ml of platelet-rich plasma. In addition, Groups E2 (n = 12) and E3 (n = 12) underwent cryotherapy or vitrectomy. Group E4 (n = 13) underwent both procedures. Seven of the 13 Group 4 experimental eyes developed total retinal detachment and giant holes. None of the other groups developed more than two total retinal detachments or giant holes (P < 0.05). Light and electron microscopy showed intravitreal or preretinal proliferation composed of fibroblast-like cells. Retroretinal membranes appeared only in Group E4 eyes, composed of elongated cells with oval nuclei and abundant organelles in the cytoplasm. We believe these lesions mimic human PVR more closely than other models previously developed.


Tear protein G originates from denatured tear specific prealbumin as revealed by two-dimensional electrophoresis.

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS PAGE) of the non-denatured low molecular weight (MW) tear proteins (dilution in phosphate buffered saline or in the non-ionic detergent Triton X100) revealed no protein G but a strongly marked 23-kD related to a tear specific prealbumin (TSP) subunit coming with the known 15, 17, 18 and 20-kD TSP subunits. Under mild denaturating conditions of sample preparation with SDS dilution just before electrophoresis, 23-kD protein decreased and a faint 32-kD protein G appeared. Under stronger denaturing conditions of sample preparation with SDS treatment (boiling or freeze-thaw cycles), 23-kD protein disappeared and two main protein G forms (32 and 34-kD) and additional bands (29, 36, 39, 42, 57 and 60-kD) appeared depending on the sample treatment. The isoelectric pH (pI) of these proteins ranged from pH 5.2 to pH 5.4. Different two-dimensional electrophoresis methods revealed that: - in presence of SDS, 23-kD protein was spontaneously changed into 17-kD TSP and such a phenomenon was partially reversible by using a non-ionic detergent (Triton X100), new proteins appeared under denaturating processes were related to various protein G forms and originated from TSP group, proteins G were produced by the aggregation of TSP subunit related to MW 17-kD/pI 5.0 corresponding to the major TSP subunit, disulfide bond formation was shown to play a major role in the aggregation process although protein G group was not totally reduced by dithiothreitol. Such results suggest that protein G is an in vitro experimental artifact due to denaturing conditions with SDS treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Intracamerally injected platelet activating factor (PAF) induces marked intraocular inflammatory reactions.

An inflammatory response was elicited in the rabbit eye by intracameral injection of platelet activating factor (PAF). PAF induced severe aqueous flare, corneal edema, pupillary constriction and marked biphasic changes in intraocular pressure (IOP) in a dose-dependent manner. All of the responses to PAF were inhibited by the PAF receptor antagonist, BN 52021 (20 mg/kg, i.p.). The cyclooxygenase inhibitor, indomethacin (30 mg/kg, i.p.) caused significant inhibition of the early phase PAF-induced aqueous flare, pupillary constriction and intraocular hypertension, but did not effect PAF-induced corneal edema or intraocular hypotension. NDGA (10 mg/kg, i.p.), a lipoxygenase inhibitor, did not inhibit the inflammatory effects of PAF. PAF-induced chemotactic response was evaluated by tissue chemiluminescence. Intracamerally injected PAF did not significantly increase chemiluminescence in cornea or iris-ciliary body, but intracorneal injection of PAF did cause a chemotactic response in both the conjunctiva and cornea. These data suggest that PAF may be an important mediator of intraocular inflammation and that some PAF-induced effects are prostaglandin dependent, while others may be independent of eicosanoid synthesis and release.


Ciliary body in experimental autoimmune uveitis: tissue repair and immunoreactivity of extracellular matrix substances.

Experimental autoimmune uveoretinitis was induced in female Lewis rats with bovine retinal soluble antigen (S-antigen). Tissue changes and immunoreactivities of transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), and extracellular matrix compounds in the anterior segment (ciliary body) were investigated by immunocytochemical methods. Control animals received adjuvant only. The immunized animals were sacrificed at day 0, 3, 7, 14, 20, and 30 postimmunization. Tissue changes that occurred at the peak of inflammation (day 14) included destruction of the inner basement membrane, epithelial cell loss, distortion of the ciliary stroma, and loss of epithelial basal infoldings. Ciliary body architecture was regenerated almost completely by day 30. Basement membrane laminin and collagen type IV levels did not change much during the inflammatory process. Fibronectin labeling level peaked at day 14 postimmunization. Collagen type V level was low at day 14 and elevated at day 20 and day 30. TGF-beta immunoreactivity peaked at day 14 and remained elevated thereafter. EGF labeling did not increase until day 20 and was maximal at day 30. Labeling of both growth factors was principally confinded to the stromal regions. The presence of TGF-beta and EGF in the ciliary stroma at well defined intervals suggests a coordinated effect upon the synthesis and reorganization of the extracellular matrix and possibly upon the inflammatory cell population in the anterior tissue.


Effects of 2-mercaptopropionylglycine on the development of X-ray-induced cataract in rats.

The effect of 2-mercaptopropionylglycine on the development of cataract induced by a single dose of X-ray (10 Gy) was investigated in rats. Intraperitoneal injection of 20 mg/kg, three times weekly starting 1 day after irradiation delayed the development of X-ray-induced cataracts significantly. The amounts of non-protein SH groups, malondialdehyde and the Na+/K+ ratio, in the lenses of rats post-treated with the drug were significantly maintained at normal levels even at 27 weeks after irradiation. On the other hand, a single administration of 250 mg/kg of the compound, 30 min prior to irradiation had no effect on cataract progression induced by X-ray.


Proteolytic activation of corneal matrix metalloproteinase by Pseudomonas aeruginosa elastase.

Purified Pseudomonas aeruginosa elastase cleaved a 65 kDa gelatinase [inactive proenzyme form of matrix metalloproteinase (MMP-2)] from human corneal fibroblasts into a biologically active fragment with an approximate molecular mass of 58 kDa. However, purified pseudomonal alkaline protease did not cleave MMP-2 appreciably. Since activated MMP-2 is known to degrade native type IV, V and VII collagens, all components of the corneal basement membrane or stroma, our results suggest a new role for pseudomonal elastase in the pathogenesis of corneal infection, inflammation and ulceration.


Methicillin-resistant Staphylococcus aureus keratitis in the rabbit: therapy with ciprofloxacin, vancomycin and cefazolin.

To determine the efficacy of a fluoroquinolone antibiotic in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) keratitis, topical administration of 0.3% ciprofloxacin was compared with topical 5.0% vancomycin or 5.0% cefazolin in experimental infections in the rabbit eye. The infections were established by intrastromal injection of 100 colony forming units (CFU) of MRSA, which resulted in greater than 10(6) CFU per cornea by 12 hr postinfection. Chemotherapy (one drop every 15 min) was given from 4-9, 10-15, or 10-20 hr postinfection. Early therapy (4-9 hr postinfection) with ciprofloxacin rendered all eyes free of bacteria; ciprofloxacin was significantly more effective than vancomycin or cefazolin. When treatment was initiated 6 hr later (10-15 hr postinfection), no corneas became free of bacteria, but ciprofloxacin was again more effective than vancomycin or cefazolin. Bacterial killing by ciprofloxacin after treatment from 10-20 hr postinfection was also significantly greater than that of vancomycin. Overall, the results show that ciprofloxacin is effective in killing methicillin-resistant staphylococcus aureus, and is most effective when applied during the very early stages of infection.


Mitotic regulation in Aspergillus nidulans.

The nimA and bimE genes of Aspergillus nidulans respectively encode a 79 kDa protein kinase that is a positive regulator of mitosis and a 229 kDa protein that is a negative regulator of mitosis. Either overproduction of nimA or inactivation of bimE can induce mitosis and override the checkpoint associated with incomplete DNA replication. Double mutants between temperature-sensitive nimA and bimE alleles undergo chromatin condensation and spindle polymerization at restrictive temperature, suggesting that the p79nimA kinase is not required for chromatin condensation and spindle polymerization when bimE function is defective. In contrast double mutants carrying ts bimE and nimEcyclinB mutations or bimE and nimTcdc25 mutations are blocked in interphase at restrictive temperature. These results indicate that the mitotic block caused by inactivation of bimE requires activation of the p34cdc2/MPF kinase for chromatin condensation and spindle polymerization to occur. Antibodies against bimE fusion proteins have been used to study p229bimE in wild-type cells and cells overexpressing the bimE gene.


DNA replication and progression through the cell cycle.

Somatic cells possess control mechanisms which monitor DNA replication and assure that it is complete before mitosis is initiated. We have been investigating these mechanisms in Xenopus egg extracts. Using in vitro cycling extracts, which spontaneously alternate between interphase and mitosis, we found that the onset of mitosis is inhibited by the presence of unreplicated DNA, demonstrating that the completion of DNA replication and the initiation of mitosis are coupled in these extracts. As in somatic cells, this coupling is sensitive to caffeine and to okadaic acid. In Xenopus extracts unreplicated DNA increases the tyrosine phosphorylation of p34cdc2, thereby maintaining MPF (mitosis-promoting factor) in an inactive state and preventing the onset of mitosis. The block to mitosis in the presence of unreplicated DNA can be reversed by the addition of bacterially expressed cdc25 protein. The extent of MPF activation by cdc25 protein under these conditions depends on the number of nuclei present. We have developed an assay to examine the rate of tyrosine phosphorylation on p34cdc2. It is increased by unreplicated DNA, in a manner consistent with unreplicated DNA up-regulating the kinase that phosphorylates p34cdc2. We have begun to examine how unreplicated DNA generates the signal that inhibits MPF activation by testing the ability of naked single- and double-stranded DNA templates to inhibit mitosis, and by investigating the role of RCC1, a chromatin-associated protein required for the coupling of DNA replication and mitosis.


Cyclins A and B1 in the human cell cycle.

Cyclins are a family of proteins involved in the regulation of the eukaryotic cell cycle. The first cyclins to be isolated were the A- and B-type cyclins and we have been studying their behaviour in human somatic cells. The levels of both cyclin A and B1 are regulated transcriptionally as well as post-translationally; both are rapidly and specifically degraded in mitosis. Cyclin A synthesis commences at the start of S phase and the protein is predominantly nuclear, whereas cyclin B1 appears during S phase and is primarily cytoplasmic. Cyclin B1 moves into the nucleus just at the start of mitosis and associates with condensed chromosomes and the mitotic spindle. Both cyclin A and cyclin B1 bind to and activate a protein serine/threonine kinase subunit; cyclin A associates with p33cdk2 and with p34cdc2, whereas cyclin B1 seems to bind exclusively to p34cdc2. Cyclin A-associated kinase activity appears much earlier in the cell cycle than that of cyclin B1, which appears only at the G2 to M transition. Therefore cyclin A may play a role in the events of S phase as well as G2 and M phases. Cyclin A forms a cell cycle-dependent complex with p33cdk2 and the transcription factor E2F, although the function of this complex is not yet clear. We conclude that cyclins A and B1 may differentially regulate the cell cycle in several ways. They form complexes with distinct protein kinases and these complexes are active at different times in the cell cycle; they form distinct multiprotein complexes, such as with the transcription factor E2F; and they are localized to different parts of the cell where different substrates will be available to them.


Is START a switch?

The cell cycle in Saccharomyces cerevisiae is controlled by regulation of START in late G1. The CLN1, CLN2 and CLN3 family of cyclin homologues is required for cells to pass START. They probably act by activating the CDC28 protein kinase. Expression of CLN1 or CLN3 under the control of an inducible promoter shows that transcription of either gene is sufficient for cyclin-deficient strains arrested in G1 to traverse START. A model of START regulation involves activation of CDC28 kinase by any CLN protein, leading to activation of CLN1 and CLN2 transcription in a positive feedback loop and passage through START. The cell cycle-dependent transcriptional regulators SWI4 and SWI6 may be components of the feedback loop. Cell cycle commitment entails resistance to the inhibitory action of mating factor, which correlates with peak levels of CLN1 and CLN2 mRNAs. FAR1 encodes an alpha-factor-dependent inhibitor of CLN function whose expression is markedly reduced at the time of START. The interplay of all these factors may sharpen the START transition such that it is close to an all-or-nothing switch event. This may be important for several START-dependent events to be activated at the same time, leading to coordinated cell cycle progression.


Regulation of CYL/cyclin D genes by colony-stimulating factor 1.

The proliferative effects of colony-stimulating factor 1 (CSF-1) on macrophages are exerted only throughout the G1 phase of the cell cycle. Genetic targets of the delayed early response to CSF-1 include novel G1 cyclin (CYL or cyclin D) genes. In macrophages, cyclin D1 is induced early in G1 and is expressed throughout the cell cycle as long as CSF-1 is present. The cyclin D1 protein turns over rapidly in CSF-1-stimulated cells and its level declines precipitously upon CSF-1 withdrawal. Cyclin D2 is induced later in G1 and its expression is periodic, whereas cyclin D3 is not expressed in macrophages but is regulated by growth factors in other cell types. The cyclin D1 protein associates during G1 with a polypeptide antigenically related to p34cdc2 and binds in vitro to a histone H1 kinase present in lysates of CSF-1-starved macrophages. The instability of the cyclin D1 protein and its ability to rescue a cyclin-dependent kinase activity from growth factor-deprived macrophages together suggest that the cyclin D protein is the dynamic partner in the complex. The timing of expression of cyclin D genes suggests that they act to link growth factor signals with cell cycle transitions during G1.


Cell cycle regulation of retinoblastoma protein phosphorylation.

The product of the retinoblastoma susceptibility gene (Rb) is a substrate of the cell cycle-regulated cdc2 and cdk kinases. The Rb protein is phosphorylated from S through M phases of the cell cycle and is dephosphorylated in G1. In in vivo phosphorylated Rb protein, we have found ten phosphotryptic peptides, all of which could be phosphorylated by cdc2 kinase, p34cdc2, in vitro. The sites of phosphorylation for eight of the ten peptides have been mapped and they conform to the known p34cdc2 phosphorylation consensus. Although the activated p34cdc2 in mitotic cells is the major phosphorylation enzyme for Rb, the Rb kinase activity of p34cdc2 is not activated at G1/S transition. A cyclin A/p33 complex is activated at G1/S. We have assembled active cyclin B1/p34cdc2 complex in insect cells. The insect cell-derived kinase complex phosphorylates histone H1 well but exhibits a poor Rb kinase activity. These results indicate that the retinoblastoma protein is phosphorylated by distinct cyclin/kinase complexes in the cell cycle and suggest a regulation of the substrate specificity of the p34cdc2/cyclin complex.


cdc2 protein kinase: structure-function relationships.

Activation of the cdc2 kinase in the cell cycle occurs upon binding to a regulatory subunit called cyclin. Cyclin A associates with both Cdc2 and its homologue Cdk2. The two complexes appear in S phase but cyclin A/Cdk2 is activated earlier than cyclin A/Cdc2. Several regions in Cdc2 are involved in binding cyclins A and B. Phosphorylation of cyclin/Cdk complexes ensures that the kinase activity peaks at a specific time in the cell cycle. Phosphorylation of Thr161 in Cdc2 is required for strong cyclin binding and kinase activity in vitro; its dephosphorylation is necessary for cells to exit mitosis. We have identified a novel 'Activating factor' that stimulates binding between cyclin and Cdc2 by inducing phosphorylation of Cdc2 on Thr161. We propose that Thr161 is targeted by an additional cell cycle regulatory pathway.


Activation of MPF in fission yeast.

In fission yeast p34cdc2/cyclin is activated at the G2/M boundary by dephosphorylation of Tyr15 of the p34cdc2 subunit. Two protein phosphatases carry out this dephosphorylation event. The major activity is encoded by cdc25, which is a distantly related member of the protein tyrosine phosphatase family. A minor activity is provided by a newly identified fission yeast protein tyrosine phosphatase.


G1 control in yeast and animal cells.

In budding yeast, Saccharomyces cerevisiae, the cell cycle is controlled at the G1/S phase transition by regulating the activity of the CDC28 protein kinase. This is the budding yeast homologue of the cdc2 protein kinase associated in most organisms with control of mitosis. In budding yeast CDC28 controls both the G1/S phase transition and the G2/M phase transition by being differentially activated by two distinct classes of positive regulatory subunits known as G1 cyclins or CLNs and B-type cyclins or CLBs, respectively. To establish whether a similar dual role for Cdc2-related kinases exists in animal cells, we and others have sought human homologues of yeast G1 cyclins. Of several candidates, cyclin E is the most promising in that it accumulates prior to S phase and is associated with a pre-S phase protein kinase activity. The kinetics of accumulation of cyclin E-associated protein kinase activity is consistent with a role at the mammalian cell cycle restriction point.


Regulation of p34cdc2 protein kinase activity by phosphorylation and cyclin binding.

Activation of the protein kinase p34cdc2 is required for entry into meiotic or mitotic M phase in all eukaryotic cells. One important mechanism regulating the activity of p34cdc2 during the cell cycle is based on phosphorylation/dephosphorylation. Avian p34cdc2 is phosphorylated on threonine 14 (Thr14), tyrosine 15 (Tyr15), threonine 161 (Thr161) and serine 277 (Ser277). Dephosphorylation of both Thr14 and Tyr15 is required for activation of p34cdc2 at the G2/M transition, indicating that phosphorylation of these residues negatively regulates p34cdc2 activity. Conversely, phosphorylation of Thr161 is required for kinase activity. Whether modification of this residue is due to intramolecular autophosphorylation or to the action of an as yet unidentified kinase remains unresolved. Likewise, the role of phosphorylation of p34cdc2 on Ser277 during G1 phase of the cell cycle remains to be determined. The function of p34cdc2 is regulated also by cell cycle-dependent complex formation with cyclin proteins. We found that chicken cyclin B2 undergoes a striking redistribution from the cytoplasm to the nucleus just prior to the onset of mitosis. Expression of a non-destructible cyclin B2 mutant causes HeLa cells to arrest in mitosis. Frequently, arrested cells displayed multiple mitotic spindles.


Cyclins and cdc2 kinases in Drosophila: genetic analyses in a higher eukaryote.

Cyclin proteins and the kinases with which they associate are encoded by gene families in multicellular eukaryotes. A variety of cyclin/kinase complexes with different functions may exist. We have started a genetic dissection of this complexity in Drosophila. We have done experiments to investigate a potential functional overlap between two kinases (Dmcdc2 and Dmcdc2c) and two cyclins (cyclin A and cyclin B). No functional overlap was observed between the Dmcdc2 and the Dmcdc2c kinases. The phenotype resulting from mutations in Dmcdc2 was not affected by altering the level of Dmcdc2c. Our results concerning cyclin A and cyclin B strongly suggest that these two cyclins have largely overlapping functions. Cell proliferation was observed in the absence of either cyclin A or cyclin B, but not if both cyclins were absent. Cyclin A also has essential functions that cannot be taken over by cyclin B, but these functions appear to be required at defined developmental stages in specific tissues only.


Age-related differences in vascular effects of pentoxifylline in isolated perfused ferret lungs.

We have determined the magnitude and sites of action of pentoxifylline (PTX), a methylxanthine derivative, in the adult and 3- to 4-week old ferret pulmonary circulation. Lungs of 8 ferrets, four 3- to 4-week-old and 4 adult, were isolated and perfused with blood. During perfusion, blood flow was kept constant, as were airway and left atrial pressures (6 and 8 cm H2O respectively, zone 3 conditions). In all lungs, pulmonary artery pressure was measured continuously and the circulation was partitioned into arteries, microvessels and veins, by measurement of pressures in 20-50 microns diameter subpleural arterioles and venules using the micropipette-servonulling method. Pressures were obtained in each lung during baseline, after vasoconstriction with hypoxia, and again after the infusion of PTX, 20 mg/kg, during hypoxia. We found that with hypoxia, total vascular resistance increased by approximately 90% in both adult and neonatal lungs; arterial and venous resistances increased by 100-180% in both age groups, with little change in microvascular resistance. PTX resulted in a significant decrease in total vascular resistance, due to a decrease in resistance in both arteries and veins. The decrease in resistance with PTX was greater in adult lungs (of the increase in resistance induced by hypoxia, 80% was eliminated by PTX) than in 3- to 4-week old lungs (51% elimination of tone induced by hypoxia). This difference was mainly due to a smaller reduction in arterial resistance with PTX in 3- to 4-week-old lungs. We conclude that PTX is a powerful pulmonary vasodilator in ferrets and that its effectiveness as a vasodilator depends on the age of the animal, the older animal showing greater responsiveness.


Age-related changes in calcium antagonist receptors in rabbit ureter.

(+)-[3H]PN 200-110 (a dihydropyridine calcium channel antagonist) binding sites were studied in ureters of 1-day (neonatal), 6-week (premature), 6-month (young) and 4.5- to 5-year (old) female rabbits. Specific binding of (+)-[3H]PN 200-110 to ureteral membrane particulates was saturable, reversible and of high affinity. The densities (Bmax) of (+)-[3H]PN 200-110 binding sites were 46.7 +/- 2.5, 22.6 +/- 2.0, 12.7 +/- 1.8 and 11.9 +/- 1.6 fmol/mg protein in 1-day, 6-week, 6-month and 4.5- to 5-year rabbit ureters, respectively. The affinity constants (KD) of the binding sites for (+)-[3H]PN 200-110 were similar in all groups. Calcium agonists and antagonists inhibited (+)-[3H]PN 200-110 binding to 1-day and 6-week rabbit ureters with the following rank order of Ki values: nitrendipine < nifedipine < BAY K 8644 < verapamil. There were no significant differences in Ki values between the neonatal and premature groups. The data demonstrate the presence of an age-related down-regulation of (+)-[3H]PN 200-110 binding sites in rabbit ureteral membrane particulates.


Relationship of prenatal caffeine exposure and zinc supplementation on fetal rat brain growth.

Pregnant rat dams were divided into four groups on the 3rd day of gestation. Group 1 dams were fed a 20% protein diet as controls. Dams of group 2 were fed a 20% protein diet supplemented with zinc (0.6 g ZnCl2/kg diet). Group 3 dams were fed a 20% protein diet supplemented with caffeine (2 mg/100 g body weight) and dams of group 4 were fed a 20% protein diet supplemented with both caffeine and zinc. Fetuses were surgically delivered on day 22, and brains were removed and analyzed for alkaline phosphatase activity, protein, zinc, cholesterol and DNA concentrations. Fetal brain caffeine levels, as well as maternal and fetal plasma caffeine levels, were determined in caffeine-supplemented groups. The body weight of group 4 and brain weights of groups 3 and 4 were higher than those of groups 1 and 2. Alkaline phosphatase activity of group 3 was less than that of group 1. The brain zinc concentration of group 2 was higher than in the other groups, but that of group 4 was less than that of group 1. The present study indicated that the supplementation of caffeine to the maternal diet decreased zinc levels in the fetal brain, and the addition of extra zinc to this diet did not return the zinc level to that of the control level as we had expected. In addition, the supplementation of caffeine and zinc together increased the body weights of the fetuses compared to the controls, but the addition of only one of these substances had no effect, suggesting that the combination of caffeine and zinc may have unique effects on fetal growth.


Effects of prenatal cocaine on the ventilatory response to hypoxia in newborn rabbits.

Recently, investigators have reported an alteration of postnatal respiratory pattern, deficient hypoxic arousal from sleep, and an increased incidence of sudden infant death syndrome (SIDS) among human infants exposed to cocaine prenatally, thus suggesting that prenatal cocaine exposure may perturb the maturation of respiratory control thereby increasing the risk for SIDS. To investigate the effects of prenatal cocaine on postnatal respiration, we evaluated the ventilatory response to 0.21 FIO2 (baseline) and at 0.15, 0.10, and 0.08 FIO2 by the barometric method on days 4-5 of life in 23 New Zealand White rabbit pups born to cocaine-exposed (30 mg/kg/day of cocaine HCl by continuous subcutaneous infusion), pair-fed and free-fed does. The chamber pressure deflection (proportional to VT after appropriate calculation) was computer-sampled at 200 Hz when the unanesthetized pups were resting quietly with no gross body movements. Recording was made after 10 min acclimatization to a specific FIO2. We found that baseline ventilation did not differ significantly among study groups. However, minute ventilation (VI), inspiratory flow (VT/TI), tidal volume (VT), increased significantly with hypoxia to peak values at 0.08 FIO2 in pair-fed and free-fed pups but these measurements did not increase significantly in cocaine-exposed pups. Our finding of a deficient second phase of the hypoxic ventilatory response among cocaine-exposed pups supports the hypothesis that prenatal cocaine perturbs the maturation of respiratory control.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of betamethasone and thyroid hormone on fetal rat lung maturation in vivo.

We studied the effect of maternal administration at various intervals of betamethasone, triiodothyronine (T3), or both, on fetal rat lung maturation. T3 alone did not enhance choline incorporation to phosphatidylcholine by 20-day fetal lung explants, or morphometric lung maturation. Betamethasone, and betamethasone plus T3, increased both of those parameters over control and T3 values. However, addition of T3 offered no advantage over administration of betamethasone alone. Significant enhancement of morphometric lung maturation was already present after only 24 h of exposure to beta-methasone, or to the combination of hormones. However, choline incorporation to phosphatidylcholine only increased significantly by 36 h of exposure to betamethasone with or without T3.


Pharmacodynamics and pharmacokinetics of cyclosporin A in the newborn pig.

The disposition kinetics of cyclosporin A in the neonates as well as age-related differences in lymphocyte responses to cyclosporin A are unknown. A single intravenous infusion of cyclosporin A was given to neonatal (2.5 or 5 mg/kg) and mature pigs (10 mg/kg) and blood cyclosporin A levels were measured by RIA. The neonates had longer elimination half-life and lower drug clearance than mature animals. Suppression in lymphocyte proliferation was only observed in mixed lymphocyte reaction and phytohemagglutinin-stimulated cultures of the 2-hour samples from neonates receiving 5 mg/kg. We conclude that neonatal pig exhibit different cyclosporin A pharmacokinetics and show higher sensitivity to cyclosporin A than mature animals.


Effect of prazosin on hypoxemia-induced blood flow redistribution in the newborn piglet.

We examined the effect of the alpha 1-adrenergic antagonist prazosin on blood pressure, left ventricular output and blood flow redistribution during normoxemia and mild hypoxemia in the chronically instrumented, unanesthetized newborn piglet employing the radiolabeled microsphere technique. Prior to prazosin, hypoxemia caused increases in aortic pressure and blood flows to the brain, myocardium and diaphragm, accomplished by small, statistically insignificant decreases in flows to the carcass and viscera without an increase in cardiac index. Prazosin treatment during normoxemia caused a fall in blood pressure and resulted in greater blood flows of left ventricular origin to the carcass, myocardium and lung. Hypoxemia after prazosin administration increased not only aortic pressure and blood flows to the brain, myocardium and diaphragm, but also, unlike the situation before drug treatment, cardiac index. Thus, in the newborn piglet, the maintenance of critical organ oxygen delivery during hypoxemia is not blocked by prazosin, but is accomplished by an increase in cardiac index rather than simply by redistribution of blood flow.


Quantitative contribution of endogenous compounds and hypoalbuminemia in reducing the binding of furosemide in the plasma of newborn infants.

The protein binding of furosemide was studied in the plasma of newborn infants and adult subjects. Plasma consisted of two pools obtained from 25 newborns and adult subjects. The concentrations of albumin were 36.8 (newborn) and 48.3 g/l (adult). The unbound fraction of furosemide was 1.38 +/- 0.15 (adult) and 2.03 +/- 0.13% (newborn; p < 0.001). After extensive dialysis of the plasma, the unbound fraction of furosemide was 1.12 +/- 0.15 (adult) and 1.39 +/- 0.09% (newborn; p < 0.0001), suggesting that dialyzable endogenous compounds interfere with the binding of furosemide. The addition of human albumin to the newborn plasma to give a final albumin concentration of 48.3 g/l yielded an unbound fraction of furosemide of 1.63 +/- 0.08 (nondialyzed plasma) and 1.17 +/- 0.08% (dialyzed plasma; p < 0.0001). The addition of albumin to the dialyzed newborn plasma, to give a final albumin concentration similar to that in adult plasma, decreased the unbound furosemide to the level of the dialyzed adult plasma. The binding defect of furosemide in newborn plasma reflects either the effects of the endogenous inhibitors or of hypoalbuminemia. The intrinsic binding properties for furosemide of newborn dialyzed plasma are similar to those of dialyzed adult plasma. This consideration corroborates our previous results on the binding of furosemide and diazepam, salicylic acid and digitoxin to newborn and adult albumin. The displacement of furosemide by salicylic acid, tolbutamide and azapropazone is 70% greater in newborn than in adult plasma. The greater displacing effect is largely due to hypoalbuminemia.


Ontogeny of vascular smooth muscle responsiveness in the postweaning rat.

This study investigated the ontogeny of contractile and relaxation responses in aortic and tail artery preparations from 3-, 7-, and 11-week-old male Sprague-Dawley rats. Contractile responses to norepinephrine, serotonin, KCl, electrical stimulation, and potassium-free physiological solution were significantly increased in vascular smooth muscle from 3-week-old rats when compared to 7- and 11-week-old rats. Endothelium-dependent acetylcholine-induced relaxation and beta-adrenoceptor mediated isoproterenol-induced relaxation were significantly attenuated with maturation. These data demonstrate that significant changes occur in aortic and tail artery smooth muscle responsiveness during the postweaning maturational period of the rat. The alterations may have significant implications with regard to cardiovascular and thermoregulatory function as well as the age of the animal when utilized as an experimental model for identifying pathogenic mechanisms involved in various disease states such as hypertension. As such, further studies are warranted to determine if similar ontogenic changes in vascular function occur at the level of the resistance vessel.


Benzoyl-CoA ligase activity in the liver and kidney cortex of weanling guinea pigs treated with various inducers: relationship with hippurate synthesis and carnitine levels.

Recent reports have indicated that guinea pig may be a better model for the study of benzoate conjugation in humans because of the presence of a salicylate-activating ligase. We gave intraperitoneal injections of sodium benzoate, sodium salicylate, sodium methylprednisolone and sodium phenobarbital for 4 consecutive days to groups of weanling guinea pigs before sacrifice. A measurement of benzoyl-CoA ligase activity and in vitro hippurate synthesis was done in liver and kidney cortex homogenates from each treated group at 21 days of age, as compared to an untreated (saline) 21-day control group. The results indicate that benzoyl-CoA ligase in guinea pigs is affected by phenobarbital (liver and kidney; p < 0.05-0.01) and methylprednisolone (liver; p < 0.01). Liver hippurate synthesis was also significantly induced by phenobarbital (p < 0.01). Both these treated groups also had reduced levels of free carnitine in plasma taken from 21-day-old guinea pigs as compared to the untreated control. Sodium benzoate did not have any effect on benzoyl-CoA ligase activity or on in vitro hippurate synthesis. However, sodium salicylate did show some effect on the induction of liver and kidney benzoyl-CoA ligase (not significant) and liver (p < 0.05) and kidney hippurate synthesis. It is important that studies on the guinea pig model be extended for application to children receiving therapy of benzoate and other aromatic acids like p-aminobenzoate or salicylate.


Effects of nimodipine on brain blood flow following acute brain ischemia in the newborn piglet.

We examined the effect of the calcium channel blocker nimodipine on postischemic hypoperfusion in the newborn piglet brain. A severe pneumothorax (SP) was induced by injecting air into the right thorax until the mean arterial blood pressure fell to 25% of baseline and was maintained for 4 min. Blood flow was immediately reduced 70-90% from baseline in each brain region during SP. In untreated animals postischemic hypoperfusion existed at 60 min, following recovery from SP with regional brain blood flow reduced 20-30% from baseline. Nimodipine infusion after SP prevented postischemic hypoperfusion in all brain regions and increased blood flows by as much as 40% above baseline in midbrain and brainstem structures. Nimodipine infusion began after severe brain ischemia prevented postischemic hypoperfusion and enhanced brain blood flow in this model.


Potential of population pharmacokinetics to reduce the frequency of blood sampling required for estimating kinetic parameters in neonates.

Data obtained from neonates receiving zidovudine as part of a phase I study were used to estimate the population pharmacokinetic parameters of this drug and to determine the minimum number of data points necessary to provide accurate estimates of the kinetic parameters and their variability. Analysis was performed with 541 concentrations of zidovudine, obtained from 32 infants and with a variety of reduced data sets using NONMEM (nonlinear mixed effect model). The reduced data sets were derived by randomly reducing the number of sampling time points per dosing interval and/or by randomly reducing the number of available subjects. We determined that accurate estimates of pharmacokinetic parameters and their variability were obtained with the inclusion of all 32 patients using only two concentration-time points per dose interval, provided that one of the points was obtained during the first 2 h after administration of the drug. The parameters themselves were adequately estimated with only 24 subjects and two concentration-time points per dose interval. We suggest that NONMEM should be used in addition to the traditional pharmacokinetic analysis to obtain more precise information directly in the population of interest with a minimum of blood sampling from each patient. This is especially critical in infants whose blood volumes are limited.


7-Ethoxycoumarin metabolism in hepatocytes from pre- and postpubescent male rats.

Marked changes in rates of drug metabolism occur during adolescence; however, biochemical events underlying alterations in drug metabolism in whole hepatocytes during this period of development are not well established. Accordingly, metabolism of 7-ethoxycoumarin, a model substrate for mixed-function oxidation, was studied in hepatocytes isolated from prepubescent and postpubescent male rats. Rates of 7-ethoxycoumarin O-deethylation increased 2.4-fold from 65 to 154 pmol/10(6) cells/min in intact hepatocytes during the narrow period of adolescence. In contrast, microsomal 7-ethoxycoumarin O-deethylase was the same in preparations from the two groups of animals. 7-Hydroxycoumarin glucuronide production in hepatocytes increased 2-fold and sulfate formation increased 16-fold across puberty. The results indicate that increases in drug metabolism, particularly sulfate conjugation, are mediated by biochemical events in addition to increases in total amounts and specific activities of hepatic drug-metabolizing enzymes.


Placental transport of dioxins from mother to fetus. II. PCBs, dioxins and furans and vitamin K metabolism.

Placental transport of dioxins and furans from mother to fetus takes place. It is probably related to the fatty acid transport. Between 10 and 20% of fatty acids in a full-term baby are of maternal origin. In adipose tissue of children that died in the early neonatal period concentrations of +/- 25% were found of three dioxin and furan congeners 12378 P5CDD, 123678 H6CDD, and 23478 P5CDF in relation to a mean concentration of these congeners in the fat of 14 breastmilk samples. Data of concentrations are given as measured in liver and adipose tissue. In the placenta of a Dutch woman an accumulation of dioxins and furans is found in relation to blood. Animal studies support the hypothesis that polychlorobifenyls play a role in the cause of the late hemorrhagic disease in the newborn, in particular the 2, 4, 5, 2, 4, 5-hexachlorobifenyl that is present in relatively high concentrations in breastmilk.


Cataract surgery and IOL implantation. More than 40 years of personal experience. My present criteria and considerations.

The author refers his personal experience with different types of IOL since 1950. After an initial period of enthusiasm (1950 to 1961) the author abandoned the techniques of Ridley, Strampelli and Dannheim, because of the considerable incidence of severe complications directly related to the IOL. Over many years he emphasized the serious postoperative complications which appeared in the course of the years, even with the different new types of IOL which acquired a certain prestige. With the new posterior chamber lenses placed in the capsular bag, the results became more encouraging and the author resumed IOL implantation with this type of lenses after an adequate period of observation and experimentation. The present criteria, technique and results of the author are discussed together with some considerations on future research work to develop new techniques for substitution of the opacified lens and also to attempt to restore accommodation.


Capsulorhexis in mature cataracts: why not?

Continuous circular capsulorhexis is now widely used in cataract surgery. In the case of mature cataract, however, this technique may be difficult due to the poor visibility of the anterior capsule. This problem can be overcome performing capsulorhexis under air. A small amount of a high molecular weight viscoelastic agent is injected near the incision, so as to avoid air escaping from the anterior chamber. Visibility is good and the handling of the anterior capsule is easy and safe.


An update on multifocal lens implants.

The performance of multifocals is partly determined by the degree to which contrast at the focussed image is reduced by out of focus images. This paper discusses how optical performance may vary with lens design and includes consideration, with available core study results, of multifocal designs marketed by Iolab, 3M, AMO, Ioptex, Storz and Domilens. For most lenses an acuity of 20/40 and J3 can be achieved unaided in over 70% of best case eyes and J2 with the distance correction in over 80%. However the risk of disappointment tends to prevent a more widespread use of multifocals among surgeons, and an explanation is given as to possible reasons.


The effect of indomethacin, diclofenac and flurbiprofen on the maintenance of mydriasis during extracapsular cataract extraction.

Surgically induced miosis (SIM) frequently occurs during extracapsular cataract extraction (ECCE). A randomized clinical trial was performed to evaluate the effect of 3 nonsteroidal antiinflammatory drugs Indomethacin 1%, Diclofenac 0.1% and Flurbiprofen 0.03%, administered topically before ECCE, on the maintenance of mydriasis during surgery. The patients were grouped based on the type of NSAID given preoperatively in addition to the standard mydriatic agents: 46 patients in group A (Indomethacin), 40 patients in B (Diclofenac), 44 patients in C (Flurbiprofen) and 34 patients formed control group D (no NSAID was instilled). Horizontal pupillary diameter measurements were taken, using a caliper, immediately prior to surgery (step 0), after capsulotomy (step I), after expression of the lens nucleus (step II) and after irrigation-aspiration of the cortical remnants (step III). Differences in pupillary diameter between step 0 and the different surgical steps were used as indices of pupillary constrictions observed in the 4 groups. A significantly less pupillary constriction was found in groups A and C than in D between steps 0 and II (p = 0.01) and in groups A and C than in B and D between steps 0 and III (p = 0.001). Our results show that Indomethacin 1% and Flurbiprofen 0.03%, compared to Diclofenac 0.1% and no NSAID regime, are significantly more effective in maintaining mydriasis during cataract surgery.


Endothelial cell density and corneal pachometry after no-stitch, small-incision cataract surgery.

In a prospective study we used the change of central and peripheral (12 o'clock-position) corneal thickness (CT) after no-stitch small incision cataract surgery as a parameter of tissue traumatisation (33 eyes) and compared the values to a series of cases (32 eyes) with conventional 3.5 mm scleral step incision. In both groups the peripheral measurements showed a higher increase in corneal thickness than the central. After 1 month all eyes regained their central preoperative thickness. Increase in corneal thickness (delta CTc, delta CTp) after the different postoperative periods were correlated. The values of the central cornea showed no significant difference between the two groups. 1, 7 and 30 days after surgery the increase of peripheral CT was significantly higher in the no-stitch group. This fact was underlined by the clinical aspect at the slit lamp and is due to the anatomical and surgical characteristic of this procedure. One month postoperatively there was no increased endothelial cell loss in the no-stitch group (3%). No-stitch cataract surgery surgery provides a lot of intra- and postoperative advantages. The problem of increased swelling of the peripheral corneal entry seems to be a secondary one as corneal thickness decreases with time. Concerning the prospective endothelial cell loss it is mandatory to study the long term results.


Cystoid macular edema in anterior chamber lens implantation following posterior capsule rupture.

By comparing the incidence of cystoid macular edema (CME) in three groups of patients having different surgical procedures, we attempted to assess the role of vitreous loss as a risk factor for CME development. In the first group (n = 470), the surgical procedure was extracapsular cataract extraction followed by implantation of posterior chamber lens (EC-CE + PC-IOL). The second group (n = 42) had extracapsular cataract extraction which was complicated by posterior capsule rupture, and therefore anterior vitrectomy followed by implantation of anterior chamber lens had to be performed (ECCE + anterior vitrectomy + AC-IOL). In the third group (n = 22) the surgery was intracapsular cataract extraction followed by anterior chamber lens implantation (ICCE + AC-IOL). The third group was included in this follow up study to assess the role of AC-IOL as a possible causative factor for development of CME in uncomplicated cases of ICCE and AC-IOL. The difference of incidences of CME in the second and third group would therefore depend mostly on the vitreous loss. The incidence of CME diagnosed by fluorescein angiography in the first, second and third group was 1.5% (7/470), 35.7% (15/42) and 9.0% (2/22), respectively. All patients who developed CME were treated with combination of corticosteroid-antibiotic drops, dexamethasone retrobulbarly (40 mg/day) and peroral indomethacin (25 mg/day/6 weeks). This therapeutic regime resulted in only moderate improvement of visual acuity.


The WHO programme for the Prevention of Blindness and cataract in developing countries.

In 1990, the WHO Programme for the Prevention of Blindness estimated that there were 13.5 million unoperated cases of cataract in the world. More than 95% of this backlog is found in developing countries. A conservative estimate of incidence of blindness due to cataract as 1/1000 population/year demonstrates that most developing countries are still unable to provide cataract surgery to the annual load of new cases. The situation is particularly worrying in Africa, south of the Sahara, where only one out of ten cataract ever gets operated on. The WHO Programme has developed a primary health care strategy for the large-scale management of cataract. Identification of cases requiring surgery should be possible at the community level, through training of auxiliary staff. Referral for surgery at the district or province hospital level is possible in most cases, given manpower development. This implies a need for training of cataract surgeons in many developing countries. There should be one cataract surgeon per 250,000 population. Increasing surgical 'productivity' of existing ophthalmologists should be considered as well as improving management of intervention programmes.


Lens-induced glaucoma.

The crystalline lens is implicated as a causative element in producing several forms of glaucoma. Etiologically they represent a diversity in the presentation of the glaucomatous process. These conditions include glaucoma related to: lens dislocation (ectopia lentis), lens swelling (intumescent cataract), classical pupillary block, aqueous misdirection--ciliary block, phacoanaphylaxis, lens particle, and phacolytic glaucoma. The management of elevated intraocular pressure often requires altering the intraocular relationship of anatomic structures surrounding the lens or lens removal. We will review the entities that produce these lens-induced glaucomatous conditions and suggest a rational approach to their diagnosis and treatment.


The role of international non-governmental organisations in dealing with cataract blindness in developing countries.

The international non-governmental organisations (INGOs) dealing with the prevention and cure of blindness in developing countries are closely networked with each other and are an integral part of the International Agency for the Prevention of Blindness (IAPE) and the World Health Organization (WHO) Global Programme for the Prevention of Blindness. As operable cataract accounts for more than half of world blindness. As operable cataract accounts for more than half of world blindness the INGOs have a particular interest in this blinding condition which took on major proportions with the eye camp movement in South Asia from the end of the 1960s. Despite this initiative, and the use of paramedic cataract surgeons in many African countries, the volume of cataract surgery is being outstripped by increased incidence arising from greater longevity as a product of improved general health. Among the options facing the INGOs in the 1991s are the increased utilisation of eye beds through year round surgery in South Asia and shorter post-operative hospital occupancy and the possibility of putting lens implantation surgery within the economic reach of INGOs and partner NGOs and governments.


The role of Western ophthalmologists in dealing with cataract blindness in developing countries.

Of the estimated 35-40 million blindness worldwide, 17 million is caused by cataract and this proportion will double by year 2025. In Africa alone, it is estimated that there are about 3 million cataract blind. As life expectancy in Africa increases so will the incidence of cataract blindness. Existing resources of manpower and services cannot cope adequately with the present backlog of cataracts, let alone the anticipated increased load. The deployment of local ophthalmologists to deal with cataract load would be cost-effective in delivering appropriate eye care but the scarcity of ophthalmologists makes the option ineffective. The role of Western ophthalmologists will be considered in the provision of cataract surgical services and training of ophthalmic assistants, cataract surgeons and ophthalmologists.


Synergistic principles of development: overlapping patterning systems in Xenopus mesoderm induction.

The first inductive event in Xenopus development establishes the mesoderm at the equator of the developing embryo. As part of this process, the dorsal-ventral and anterior-posterior axes of the embryo are initially established. A number of signalling molecules which may play a role in mesodermal induction and patterning have been identified in the last several years, including members of the FGF, TGF-beta and Wnt gene families. A variety of experiments, using either purified factors or injection of RNA encoding these factors, have added to the wealth of classical embryological experimental data collected over the last century. We have synthesized some recent results with the classical data to provide a framework for examining the process of mesoderm induction, and to formulate putative roles for some of the different factors. We incorporate these ideas into a working model of mesoderm induction that provides a basis for future experimental directions. Finally, we suggest that mesoderm induction may not be a discrete set of well separated events, but instead may be a process involving partially overlapping signals that produce the same pattern.


Expression of en and wg in the embryonic head and brain of Drosophila indicates a refolded band of seven segment remnants.

Based on the expression pattern of the segment polarity genes engrailed and wingless during the embryonic development of the larval head, we found evidence that the head of Drosophila consists of remnants of seven segments (4 pregnathal and 3 gnathal) all of which contribute cells to neuromeres in the central nervous system. Until completion of germ band retraction, the four pregnathal segment remnants and their corresponding neuromeres become arranged in an S-shape. We discuss published evidence for seven head segments and morphogenetic movements during head formation in various insects (and crustaceans).


Cleavage and gastrulation in the shrimp Sicyonia ingentis: invagination is accompanied by oriented cell division.

Embryos of the penaeoidean shrimp Sicyonia ingentis were examined at intervals during cleavage and gastrulation using antibodies to beta-tubulin and DNA and laser scanning confocal microscopy. Cleavage occurred in a regular pattern within four domains corresponding to the 4-cell-stage blastomeres and resulted in two interlocking bands of cells, each with similar spindle orientations, around a central blastocoel. Right-left asymmetry was evident at the 32-cell-stage, and mirror-image embryos occurred in a 50:50 ratio. Gastrulation was initiated by invagination into the blastocoel at the 62-cell-stage of two mesendoderm cells, which arrested at the 32-cell-stage. Further invagination and expansion of the archenteron during gastrulation was accompanied by rapid and oriented cell division. The archenteron was composed of presumptive naupliar mesoderm and the blastopore was located at the site of the future anus of the nauplius larva. In order to trace cell lineages and determine axial relationships, single 2- and 4-cell-stage blastomeres were microinjected with rhodamine-dextran. The results showed that the mesendoderm cells which initiated gastrulation were derived from the vegetal 2-cell-stage blastomere, which could be distinguished by its slightly larger size and the location of the polar bodies. The mesendoderm cells descended from a single vegetal blastomere of the 4-cell-stage. This investigation provides the first evidence for oriented cell division during gastrulation in a simple invertebrate system. Oriented cell division has previously been discounted as a potential morphogenetic force, and may be a common mechanism of invagination in embryos that begin gastrulation with a relatively small number of cells.


Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.

During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.


Embryonic alkaline phosphatase is expressed at M-phase in the spermatogenic lineage of the mouse.

We have recently cloned and characterized a novel embryonic alkaline phosphatase (EAP) expressed at the two-cell to blastocyst stage of preimplantation development in the mouse. The isozyme is re-expressed in trace amounts in the thymus, intestine and testis during adult life. In the present report, we find that EAP transcripts can be detected, by RT-PCR analysis, in very low amounts in the testes of newborn mice, but at 24 days of age EAP mRNA levels reach the highest concentrations, remaining high at 40 and even 117 days of age. We produced a synthetic peptide and a corresponding rabbit anti-peptide antiserum (Rb-1434), which was characterized by enzyme antigen immunoassays and reactivity with chinese hamster ovary cell transfectants, as reacting specifically with EAP. The Rb-1434 antibody enabled us to examine immunohistochemically what cell types in the testis are responsible for the expression of EAP during different developmental stages. No positive cells were recognized in the testis of newborns (day 0) and 8-day-old mice. Positive cells were first observed at day 15 and, at 24 days of age, many positive M-phase cells, morphologically corresponding to spermatocytes in mid to late prophase of meiotic division I, were strongly positive for EAP expression. Positive M-phase cells were also observed at 40 days and 151 days of age. Transgenic mice expressing the human GCAP isozyme in a tissue-specific manner in the testis, showed equivalent stages of M-phase figures when stained immunohistochemically with a specific rabbit polyclonal antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)


A transgenic model for studying development of the enteric nervous system in normal and aganglionic mice.

The dopamine beta-hydroxylase promoter has been shown to direct expression of the reporter gene product, beta-galactosidase, to enteric neurons and putative embryonic neuroblasts in transgenic mice (Mercer et al., 1991; Kapur et al., 1991). In this paper, expression of the transgene, D beta H-nlacZ, in the gastrointestinal tract is characterized in more detail in wild-type mice and mice which are also homozygous for the lethal spotted allele (ls). Expression of the transgene in wild-type embryos was first detected in scattered mesenchymal cells in the proximal foregut on embryonic day 9.5, and progressed distally until embryonic day 13.5 when the entire length of the gut was colonized by such cells. Several observations suggest that the mesenchymal cells which express the transgene (MCET) are, in fact, enteric neuroblasts, probably derived from the vagal neural crest. (1) The presence of MCET in progressively more caudal portions of the embryonic gut correlated with the neurogenic potential of isolated gastrointestinal segments grafted under the renal capsule. (2) Mitotic activity of MCET was demonstrated by incorporation of [3H]thymidine in utero. (3) The migratory behavior of MCET and/or their precursors was revealed in anastomotic subcapsular grafts of gut from transgenic and non-transgenic embryos; enteric ganglia of the latter were populated by MCET from the former. (4) Enteric expression of the transgene postnatally was restricted to intrinsic neurons that coexpressed other phenotypic markers of neuronal differentiation. The pattern of transgene expression in ls/ls mice was identical to that seen in ls/+ and +/+ mice until embryonic day 12.5.(ABSTRACT TRUNCATED AT 250 WORDS)


The neurogenic locus brainiac cooperates with the Drosophila EGF receptor to establish the ovarian follicle and to determine its dorsal-ventral polarity.

We have characterized the function of a new neurogenic locus, brainiac (brn), during oogenesis. Homozygous brn females lay eggs with fused dorsal appendages, a phenotype associated with torpedo (top) alleles of the Drosophila EGF receptor (DER) locus. By constructing double mutant females for both brn and top, we have found that brn is required for determining the dorsal-ventral polarity of the ovarian follicle. However, embryos from mature brn eggs develop a neurogenic phenotype which can be zygotically rescued if a wild-type sperm fertilizes the egg. This is the first instance of a Drosophila gene required for determination of dorsal-ventral follicle cell fates that is not required for determination of embryonic dorsal-ventral cell fates. The temperature-sensitive period for brn dorsal-ventral patterning begins at the inception of vitellogenesis. The interaction between brn and DER is also required for at least two earlier follicle cell activities which are necessary to establish the ovarian follicle. Prefollicular cells fail to migrate between each oocyte/nurse cell complex, resulting in follicles with multiple sets of oocytes and nurse cells. brn and DER function is also required for establishing and/or maintaining a continuous follicular epithelium around each oocyte/nurse cell complex. These brn functions as well as the brn requirement for determination of dorsal-ventral polarity appear to be genetically separable functions of the brn locus. Genetic mosaic experiments show that brn is required in the germline during these processes whereas the DER is required in the follicle cells. We propose that brn may be part of a germline signaling pathway differentially regulating successive DER-dependent follicle cell activities of migration, division and/or adhesion and determination during oogenesis. These experiments indicate that brn is required in both tyrosine kinase and neurogenic intercellular signaling pathways. Moreover, the functions of brn in oogenesis are distinct from those of Notch and Delta, two other neurogenic loci that are known to be required for follicular development.


Down-regulation of the POU transcription factor SCIP is an early event in oligodendrocyte differentiation in vitro.

The POU-domain transcription factor SCIP (also known as Tst-1) has been implicated in the development of Schwann cells, the myelinating cells of the peripheral nervous system (PNS). We have investigated the possibility that SCIP also might play a role in the development of oligodendrocytes, the myelinating cells of the central nervous system (CNS). We purified oligodendrocyte precursors (O-2A progenitors) by immunoselection and cultured them in the presence of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), which together keep O-2A progenitors proliferating and prevent oligodendrocyte differentiation. Under these culture conditions, O-2A progenitors expressed high levels of SCIP mRNA and protein, and did not express myelin-specific genes. When oligodendrocyte differentiation was initiated by withdrawing the growth factors, SCIP mRNA was rapidly down-regulated, followed by a decline in SCIP protein and the sequential activation of myelin-specific genes. Rapid down-regulation of SCIP mRNA required continued protein synthesis. In O-2A progenitors that were cultured in the presence of PDGF alone, SCIP expression declined to an intermediate level, and low levels of the myelin gene products were induced. Thus, the level of SCIP expression in O-2A progenitors is inversely related to the level of myelin gene expression, suggesting that SCIP may be involved in the developmental switch from proliferation to differentiation in the oligodendrocyte lineage. When O-2A progenitors are cultured in the presence of 10% fetal calf serum, they differentiate into type-2 astrocytes rather than oligodendrocytes. SCIP mRNA was also down-regulated in type-2 astrocytes, which do not express myelin genes, so down-regulation of SCIP seems to be more closely linked to the cessation of cell proliferation per se than the expression of a particular differentiated phenotype.


NeuN, a neuronal specific nuclear protein in vertebrates.

A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.


Actin filaments, stereocilia and hair cells of the bird cochlea. VI. How the number and arrangement of stereocilia are determined.

Beginning in 8-day embryos, stereocilia sprout from the apical surface of hair cells apparently at random. As the embryo continues to develop, the number of stereocilia increases. By 10 1/2 days the number is approximately the same as that encountered extending from mature hair cells at the same relative positions in the adult cochlea. Surprisingly, over the next 2-3 days the number of stereocilia continues to increase so that hair cells in a 12-day embryo have 1 1/2 to 2 times as many stereocilia as in adult hair cells. In short, there is an overshoot in stereociliary number. During the same period in which stereocilia are formed (9-12 days) the apical surface of each hair cell is filled with closely packed stereocilia; thus the surface area is proportional to the number of stereocilia present per hair cell, as if these features were coupled. The staircase begins to form in a 10-day embryo, with what will be the tallest row beginning to elongate first and gradually row after row begins to elongate by incorporation of stereocilia at the foot of the staircase. Extracellular connections or tip linkages appear as the stereocilia become incorporated into the staircase. After a diminutive staircase has formed, eg. in a 12-day embryo, the remaining stereocilia located at the foot of the staircase begin to be reabsorbed, a process that occurs during the next few days. We conclude that the hair cell determines the number of stereocilia to form by filling up the available apical surface area with stereocilia and then, by cropping back those that are not stabilized by extracellular linkages, arrives at the appropriate number. Furthermore, the stereociliary pattern, which changes from having a round cross-sectional profile to a rectangular one, is generated by these same linkages which lock the stereocilia into a precise pattern. As this pattern is established, we envision that the stereocilia flow over the apical surface until frozen in place by the formation of the cuticular plate in the apical cell cytoplasm.


Cloning and developmental expression of Sna, a murine homologue of the Drosophila snail gene.

The genetic analysis of dorsoventral patterning in Drosophila has identified a zinc-finger gene, snail, that is required for mesoderm formation. The cloning and nuclease protection analysis of a Xenopus homologue of this gene has suggested a possible role in the mesoderm of vertebrates. Here, we describe the cloning of a murine homologue of snail, Sna, and in situ hybridisation studies of its developmental expression. Sequence analysis reveals substantial conservation of the second to fifth zinc fingers, but not of the first zinc finger in the Sna gene. Expression occurs in the ectoplacental cone, parietal endoderm, embryonic and extraembryonic mesoderm, in neural crest and in condensing precartilage. Based on the timing and spatial restriction of expression in embryonic mesoderm, we suggest that Sna might be required for the early development of this tissue, as is the case for its Drosophila counterpart. In addition, we propose that Sna might have an analogous role in the development of neural crest. The expression in condensing precartilage indicates that this gene also has a later function in chondrogenesis.


Expression of four zebrafish wnt-related genes during embryogenesis.

The wnt gene family codes for a group of cysteine-rich, secreted proteins, which are differentially expressed in the developing embryo and are possibly involved in cellular communication. Here, we describe the polymerase chain reaction based cloning and embryonic expression patterns of four zebrafish wnt-related sequences; wnt[a], wnt[b], wnt[c] and wnt[d]. One of these genes, wnt[a], is a potential homologue of murine Wnt-3, while the other three genes most likely represent new members of the vertebrate wnt gene family. In zebrafish embryos, transcripts of wnt[a] are confined to the dorsal diencephalon, the dorsal midbrain, the rhombic lips and the dorsal portions of the spinal cord. wnt[b] is expressed in the tail bud and at considerably lower levels in the mesoderm of the head. wnt[c] transcripts are present within the diencephalon and the posterior midbrain whereas wnt[d] shows a surprisingly similar expression pattern to zebrafish wnt-1. By analogy to wnt-1, it is likely that the members of the zebrafish wnt gene family play an important role in cell-to-cell signalling during pattern formation in the neural tube and the tail bud.


Differential expression and regulation of two distinct fibroblast growth factor receptors during early development of the urodele amphibian Pleurodeles waltl.

Fibroblast growth factor (FGF) has been shown to be involved in mesoderm induction during amphibian development. Its presence in the embryo suggests that FGF is an endogenous inducer. By polymerase chain reaction (PCR) methods and by screening a Pleurodeles waltl tail-bud cDNA library with a cDNA probe for human FGF receptor, we have isolated two full-length cDNA clones, which we designate PFR1 and PFR4 based on their homology to the human FGF receptors FGFR-1 and FGFR-4. Both cDNA clones encode Pleurodeles FGF receptors that share characteristics common to members of the FGF receptor superfamily. The deduced amino acid sequence of PFR1 is 85% identical overall with the human fms-like-gene (FLG). PFR4 is most closely related to the human FGFR-4 (66% overall identity). The tyrosine kinase catalytic domains of both receptors are remarkably conserved. The two receptors show distinct patterns of regulation during early development. PFR1 first appears as a maternally derived mRNA and mRNA levels remain constant during early developmental stages. However, PFR4 mRNA is first expressed at the late blastula stage, which suggests that its expression is a result of zygotic transcription. Furthermore, northern blot analysis indicates that PFR1 mRNA is distributed evenly in the early gastrula while PFR4 mRNA is predominantly localized to the presumptive ectoderm. At tail-bud stage, PFR1 transcripts are localized primarily to the neural and mesodermal tissues, PFR4 transcripts are most abundantly expressed in neural tissue, and more transcripts are detected in lateral plate mesoderm than in the somites. When animal cap explants of blastulae are cultured in the presence of mesoderm-inducing factors, PFR1 mRNA levels are maintained by bFGF and activin A. In contrast, PFR4 mRNA levels are significantly down-regulated. These observations suggest a differential expression and regulation of FGF receptors in early amphibian development.


Transforming growth factor-beta control of cell-substratum adhesion during avian neural crest cell emigration in vitro.

It has been proposed that, in higher vertebrates, the onset of neural crest cell migration from the neural tube involves spatially and temporally coordinated changes in cellular adhesiveness that are under the control of external signals released in the extracellular milieu by neighboring tissues. In the present study, we have analyzed the dynamics of changes in cell-substratum adhesiveness during crest cell emigration and searched for regulatory cues using an in vitro model system. This model is based on the fact that, in vivo, crest cell dispersion occurs gradually along a rostrocaudal wave, allowing us to explant portions of the neural axis, termed migratory and premigratory levels, that differ in the time in culture at which neural crest cells initiate migration and in the locomotory behavior of the cells. We found that neural crest cell emigration is not triggered by the main extracellular matrix molecules present in the migratory pathways, as none of these molecules could abolish the intrinsic difference in the timing of emigration between the different axial levels. Using an in vitro adhesion assay, we found that presumptive neural crest cells from premigratory level explants gradually acquired the ability to respond to extracellular matrix material with time in culture, suggesting that acquisition of appropriate, functional integrin receptors was a necessary step for migration. Finally, we showed that members of the transforming growth factor-beta family reduced in a dose-dependent manner the delay of neural crest cell emigration from premigratory level explants and were able to increase significantly the substratum-adhesion properties of crest cells. Our results suggest that acquisition of substratum adhesion by presumptive neural crest cells is a key event during their dispersion from the neural tube in vitro, and that members of the transforming growth factor-beta family may act as potent inducers of crest cell emigration, possibly by increasing the substratum adhesion of the cells.


The big brain gene of Drosophila functions to control the number of neuronal precursors in the peripheral nervous system.

big brain (bib) is one of the six known zygotic neurogenic genes involved in the decision of an ectodermal cell to take on the neurogenic or the epidermogenic cell fate. Previous studies suggest that bib functions in a pathway separate from the one involving Notch and other known neurogenic genes. For a better understanding of the bib function, it is essential first to characterize the mutant phenotype in detail. Our mutant analyses show that loss of bib function approximately doubles the number of neuronal precursors and their progeny cells in the embryonic peripheral nervous system. Mosaic studies reveal a hypertrophy of sensory bristles in bib mutant patches in adult flies. Our observations are compatible with a function of bib in specifying neuronal precursors of both the embryonic and adult sensory nervous system. This is in contrast to the function of Notch, which continues to be required at multiple stages of neural development subsequent to this initial determination event.


Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay.

Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.


Planar and vertical signals in the induction and patterning of the Xenopus nervous system.

The cellular mechanisms responsible for the formation of the Xenopus nervous system have been examined in total exogastrula embryos in which the axial mesoderm appears to remain segregated from prospective neural ectoderm and in recombinates of ectoderm and mesoderm. Posterior neural tissue displaying anteroposterior pattern develops in exogastrula ectoderm. This effect may be mediated by planar signals that occur in the absence of underlying mesoderm. The formation of a posterior neural tube may depend on the notoplate, a midline ectodermal cell group which extends along the anteroposterior axis. The induction of neural structures characteristic of the forebrain and of cell types normally found in the ventral region of the posterior neural tube requires additional vertical signals from underlying axial mesoderm. Thus, the formation of the embryonic Xenopus nervous system appears to involve the cooperation of distinct planar and vertical signals derived from midline cell groups.


Pintallavis, a gene expressed in the organizer and midline cells of frog embryos: involvement in the development of the neural axis.

We have identified a novel frog gene, Pintallavis (the Catalan for lipstick), that is related to the fly fork head and rat HNF-3 genes. Pintallavis is expressed in the organizer region of gastrula embryos as a direct zygotic response to dorsal mesodermal induction. Subsequently, Pintallavis is expressed in axial midline cells of all three germ layers. In axial mesoderm expression is graded with highest levels posteriorly. Midline neural plate cells that give rise to the floor plate transiently express Pintallavis, apparently in response to induction by the notochord. Overexpression of Pintallavis perturbs the development of the neural axis, suppressing the differentiation of anterior and dorsal neural cell types but causing an expansion of the posterior neural tube. Our results suggest that Pintallavis functions in the induction and patterning of the neural axis.


Parthenogenetic stem cells in postnatal mouse chimeras.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)


Etiology and treatment of fluid retention (hydrops) in Ménière's syndrome.

For years low sodium diets have been recommended in the treatment of Ménière's syndrome. Elevated levels of insulin play an important role in sodium retention in renal tubules. Insulin production is stimulated by high carbohydrate diets. Adrenaline, cortisone, and glucagon levels may be increased by stress or food or inhalant allergies, further elevating insulin levels. The end result of prolonged hyperinsulinemia includes vasoconstriction and eventually arterial smooth muscle hypertrophy. Individual susceptibility to Ménière's syndrome may occur as a result of inflammatory changes in the endolymphatic sac or cochlear aqueduct secondary to primary or latent viral infections, thus predisposing to fluid retention. Long term medical treatment of Ménière's should be directed towards preventing sodium retention through sodium restriction and carbohydrate management. Other factors including stress and allergy should also be considered.


Lateral cervical radiographs and adenoid size: do they correlate?

Clinicians have questioned the value of lateral soft tissue neck x-ray (LSTN) in assessing adenoid size. Elaborate cephalometric assays have been devised to measure degree of nasopharyngeal obstruction secondary to adenoid hypertrophy. This study prospectively studied 73 children, aged 11 months to 13 years, with clinical evidence of adenoid hypertrophy to assess how well a LSTN correlates with direct intraoperative observation of adenoid size and nasopharyngeal obstruction. We found a relatively weak correlation (Pearson coefficient r = 0.34) between x-ray and operative observations. We conclude that LSTN is an appropriate examination in the preoperative assessment of children being considered for adenoidectomy. However, this test must be interpreted by recognizing the inherent limitations of evaluating a dynamic structure, such as the nasopharynx, through a non-dynamic modality.


A prospective study of ocular abnormalities in hearing impaired and deaf students.

In a prospective study of 505 hearing impaired and deaf students conducted at Children's Hospital Eye Clinic, 48.7% were found to have significant ocular abnormalities. The prevalence of myopia, astigmatism, and pathological intraocular changes was found to be significantly increased over the general population. In addition, the prevalence of ocular abnormalities generally increased with the severity of the hearing loss. Of importance was the high prevalence of rubella-consistent abnormalities in the hearing impaired and deaf student populations. Rubella syndrome may be responsible for a major portion of high refractive errors and ocular pathological changes. It is recommended that ENT specialists and pediatricians be aware of the nearly 50% chance of ocular abnormality in hearing impaired and deaf students, and that ophthalmologic examination may be valuable for identification or confirmation of the etiology of hearing impairment.


Contemporary skin grafting in otologic surgery.

In the post-operative healing phase following external auditory canalplasty or open cavity mastoidectomy, problematic epithelialization of surfaces may ensue. Exposed surfaces or surface epithelialized with tissues other than skin may predispose to secondary infection and result in recurrent otorrhea. Split-thickness skin grafts can be useful in promoting healing of exposed canal and mastoid cavity surfaces. Our experience and methodology for harvesting and placing these grafts are reviewed. The indications, management, and complications of split-thickness otologic skin grafting will be presented.


Neurotologic manifestations of von Hippel Lindau disease.

Von Hippel Lindau disease is a hereditary phakomatosis characterized by congenital angiomatosis of the retina and cerebellum. This autosomal dominant syndrome exhibits variable penetrance and expressivity. Because of the marked clinical variability and sporadic age of onset, members of affected families must be counseled and screening protocols established. It has been recommended that initial screening include appropriate laboratory work, ophthalmologic and neurologic examination, radiologic evaluation of the CNS, and when appropriate, abdominal ultrasound. Follow-up examination should be annually with acute evaluations as focal signs and symptoms develop. With the help of technological advances and screening of family members, patients with vHL disease can live long, productive lives with reduced morbidity.


Laryngeal chondrosarcoma: diagnosis and management.

Chondrosarcoma of the larynx is a rare malignancy accounting for 0.5% of all laryngeal tumors. We report our approach to laryngeal chondrosarcoma and review the recent literature on the subject. Our review supports the previously held opinions about sites of origin, but draws new conclusions about symptoms. In addition, we examine the controversy of conservative surgery vs. total laryngectomy.


Submandibular rhabdomyoma.

A case of recurrent adult rhabdomyoma of the submandibular gland is presented. This is a rare tumor of the skeletal muscle. Approximately 80 cases have been reported. The salient pathologic features are reviewed and a discussion and review of the literature is presented.


The hydration shell of myoglobin.

The space in the unit cell of a metmyoglobin crystal not occupied by myoglobin atoms was filled with water using Monte Carlo calculations. Independent calculations with different amounts of water have been performed. Structure factors were calculated using the water coordinates thus obtained and the known coordinates of the myoglobin atoms. A comparison with experimental structure factors showed that both the low and the high resolution regime could be well reproduced with 814 Monte Carlo water molecules per unit cell with a B-value of 50 A2. The Monte Carlo water molecules yield a smaller standard R-value (0.166) than using a homogeneous electron density for the simulation of the crystal water (R = 0.212). A reciprocal space refinement of the water and the protein coordinates has been performed. Monte Carlo calculations can be used to obtain information for crystallographically invisible parts of the unit cell and yield better coordinates for the visible part in the refinement.


Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).


Conformational change of a synthetic amyloid analogue des[Ala21,30]A42 upon binding to octyl glucoside micelles.

The secondary structure of a synthetic amyloid fragment des [Ala21,30]A42 was studied by circular dichroism and Fourier transformed infrared spectroscopy. Measurements were performed in trifluoroethanol/water and octyl beta-D-glucopyranoside solutions. The spectra of the peptide in trifluoroethanol indicate a high percentage of alpha-helical structure. However, in octyl glucoside, at and above the critical micelle concentration, the peptide adopts a beta-sheet conformation. Secondary structure analysis yields a predominant (> 70%) beta-sheet content. Our data suggest that the peptide backbone or polar side groups of des[Ala21,30]A42 interact with the sugar-coated surface of micelles, which promotes an alpha to beta conformational transition.