Cell-cell association directed mitotic spindle orientation in the early development of the marine shrimp Sicyonia ingentis

During early cleavages of Sicyonia ingentis embryos, mitotic spindle orientations differ between blastomeres and change in a predictable manner with each successive mitosis. From 2nd through 7th cleavages, spindles orient at a 90 degrees angle with respect to the spindle of the parent blastomere. Thus, spindle orientation is parallel to the cleavage plane that formed the blastomere. To determine if specific spindle orientations were intrinsic properties of individual blastomeres, we altered blastomere associations and asked how mitotic spindle orientation was affected in successive cleavages using laser scanning confocal microscopy. Linear embryos were constructed by dissociating 4-cell embryos and recombining the blastomeres in a linear array. The ensuing cleavage (3rd embryonic cleavage) of these linear embryos was parallel to the long axis of the embryo, resulting in four parallel pairs of blastomeres which lay in a common plane that was parallel to the substratum. The 4th cleavage produced a linear embryo with the 16 blastomeres arranged in four parallel quartets. Then, in preparation for 5th cleavage, spindles oriented at a 45 degrees angle (not parallel as in normal development) with respect to the previous cleavage plane. When 8-cell linear embryos were separated into linear half-embryos, subsequent spindle orientations were not like those observed for intact 8-cell linear embryos, but rather regressed to the orientation seen in 4-cell linear embryos. We suggest that the reorientation of mitotic spindles during early cleavage of S. ingentis is neither an intrinsic property nor age dependent, but rather is cell contact related. Further, these results in conjunction with observations of non-manipulated embryos suggest that spindle poles (centrosomes) avoid cytoplasmic regions adjacent to where there is cell-cell contact during early development.     

Hyperglycaemic hormones inhibit protein and mRNA synthesis in in vitro-incubated ovarian fragments of the marine shrimp Penaeus semisulcatus

The specificity of many signal transduction pathways relies on the temporal coordination of different second messenger signals. Here we found a molecular mechanism which guarantees that conventional protein kinase C (PKC) isoforms are sequentially activated by calcium and diacylglycerol signals. Receptor stimuli that triggered repetitive calcium spikes induced a parallel repetitive translocation of GFP-tagged PKCgamma to the plasma membrane. While calcium acted rapidly, diacylglycerol binding to PKCgamma was initially prevented by a pseudosubstrate clamp, which kept the diacylglycerol-binding site inaccessible and delayed calcium- and diacylglycerol-mediated kinase activation. After termination of calcium signals, bound diacylglycerol prolonged kinase activity. The properties of this molecular decoding machine make PKCgamma responsive to persistent diacylglycerol increases combined with high- but not low-frequency calcium spikes.     

Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa

It is widely agreed that after two or more seizures patients should be given antiepileptic treatment, but there is still controversy about the treatment of patients after a first unprovoked seizure. In a multicenter, randomized, open trial, patients with a first tonic-clonic seizure were randomized to immediate treatment (carbamazepine, phenytoin, phenobarbital, or sodium valproate) or to treatment only after another seizure. Fifty-two (24%) of the 215 patients randomized to immediate treatment and 85 (42%) of the 204 randomized to delayed treatment experienced seizure recurrence during follow-up. Age, acute treatment of the seizure with benzodiazepines, remote etiologic factors, and EEG abnormalities were significant predictors of relapse. Of the immediately treated patients, 87% had no seizures for a year and 68% had no seizures for 2 years, whereas only slightly fewer initially untreated patients (83% and 60%) achieved these endpoints. Patients treated after the first seizure and those treated after seizure relapse had the same time-dependent probability of achieving 1 and 2 seizure-free years. None of the variables that were prognostic predictors of relapse was significantly associated with the probability of having 1 or 2 years of seizure control. Anticonvulsants in patients presenting a first tonic-clonic seizure reduce the risk of relapse; however, 50% of patients who are not treated will never experience a second seizure. Moreover, the probability of long-term remission is not influenced by treatment of the first seizure.     

Deficiency of fucosidase results in acrosomal dysgenesis and impaired sperm maturation

The goal of therapeutic vaccination is to elicit an antiviral immune response within persistently infected individuals and consequently eradicate the infection. CD8+ T cells are potent mediators of viral clearance; however, during chronic infections CD4+ T cell help is required to sustain antiviral CD8+ T cell activity. Therapeutic vaccination should be targeted towards enhancing both CD4+ and CD8+ T cell virus-specific responses.     

Quasi- and nonequivalence in the structure of bacterial flagellar filament

In supercoiled forms of flagellar filaments, which are thought to be produced by combinations of two distinct subunit lattices, the lattices are elastically deformed in 11 different ways, depending on their azimuthal positions on the circumference of a tube with 11 protofilaments. Those two interactions are nonequivalent as opposed to quasiequivalent ones in elastically deformed lattices of otherwise identical interactions. The term nonequivalence is defined to represent different bonding interactions, and quasiequivalent is used to describe deformed but conserved bonding interactions. By using two distinct lattices that were accurately determined by x-ray fiber diffraction, 10 possible supercoiled forms of flagellar filaments were simulated, based on a bistable-subunit packing model. Comparison to the observed forms showed good agreement, indicating that the model and determined lattice parameters effectively represent realistic features of the structure. The simulated quasiequivalent lattices have been compared to the two nonequivalent lattices, revealing an interesting feature: the maximum deviation in the intersubunit distance by elastic deformation is almost three-quarters of the difference between the two distinct lattices, demonstrating a balanced coexistence of a well-defined conformational distinction and extensive adaptability in the molecular structure of flagellin and its packing interactions.     

Trypsin-like enzymes during fertilization in the shrimp Rhynchocinetes typus

OBJECTIVE: To determine factors affecting postoperative pulmonary circulation in patients with major systemic-to-pulmonary collateral arteries. METHODS: A total of 48 patients underwent biventricular repair subsequent to unifocalization at ages in the range 1-34 years. The preparative procedures consisted of ligation of the collateral arteries in 6, plasty to the pulmonary arteries using no artificial materials in 12 and extensive reconstruction using heterologous pericardial tubes in 30. The number of the pulmonary vascular segments unifocalized was 9-18 (16 +/- 3). The amount of flow draining via residual minute systemic-to-pulmonary collaterals measured at the time of repair was 4-58% (24 +/- 16%) of the total perfusion by the cardiopulmonary bypass machine. RESULTS: This value was 40 +/- 16% in 5 patients dying in the short term after repair. The number of segments was nine or ten after unifocalization in 2 of these. Another 4 patients died in the longer term, 3 of these with CATCH 22 syndrome dying because of pulmonary hypertension. Postoperative catheterization demonstrated mean pulmonary arterial pressures in the range 8-40 (21 +/- 9) mmHg and pulmonary resistance in the range 1.7-10 (5.0 +/- 2.1) units/m2. Pulmonary resistance was correlated statistically to age at repair (r = 0.77), the number of pulmonary vascular segments (r = -0.41) and to percent collateral flow (r = 0.48). The use of a heterologous pericardial tube for unifocalization was also related probably to higher pulmonary resistance. CONCLUSION: It is essential to accomplish effective unifocalizations followed by earlier definitive repair so as to establish better pulmonary circulation.     

Survey of disease of cultural shrimp in Taiwan

A water-in-oil-in-water (w/o/w) technique, sometimes known as in-water drying method, was used to prepare microcapsules consisting of polylactic acid and poly(lactide-co-glycolide). The influence of shear force to produce an initial water-in-oil (w/o) emulsion on the characteristics of microcapsules and protein release was investigated. Bovine serum albumin (BSA) was used as the model protein drug for encapsulation. The initial w/o emulsion was prepared by a Polytron homogenizer. The shear rate was varied from 11 to 23 krpm to produce w/o emulsions with different shear forces. This study revealed pronounced effects of shear force on the characteristics of microcapsules and release profiles of BSA. Depending on the degree of the shear applied, the inner structure of microcapsules showed very different morphology, which was responsible for different release patterns. A low shear produced microcapsules with a high initial burst release of BSA, whereas microcapsules using a high shear exhibited a controlled release of protein without any initial burst release. Also, at a given shear force, a variation in polymer composition of microcapsules was found to be effective in controlling the release characteristics of protein. Thus, the homogenization technique should be carefully considered in designing microcapsules with desirable release profiles of proteins and an adequate period of protein delivery.     

Phenotypic characterisation of the dendritic cell infiltrate in prostate cancer

4-Nitrocatechol was identified as a product of transformation of 4-nitrophenol by bacterial strain Corynebacterium sp.8/3 using direct acetylation of biodegradation samples by acetic anhydride followed by GC-MS analysis. The identity of 4-nitrocatechol, in the form of diacetate, was confirmed by electron-impact spectra and spectra recorded under chemical ionization conditions (positive and negative modes). Negative-ion chemical ionization was used for quantification of 4-nitrocatechol in biodegradation samples in a concentration range of 1-25 mg/l.     

Immunological problems of transplantation into the subretinal space

The objective of retinal transplantation is to substitute destroyed or degenerated retina to improve visual function. Photoreceptors and retinal pigment epithelium cells of embryonic and nonembryonic origin have been transplanted into the subretinal space in different animal models. Recently, retinal cells have also been used for transplantation in untreated or immunosuppressed patients with retinitis pigmentosa and age-related macular degeneration. Transplants performed in animals such as the RCS rat have maintained retinal function at the transplantation site for long periods of time, although such transplantations in humans have not proved conclusively, to date, whether there is a real effect or not. One reason for this phenomenon seems to be an immune response to transplanted retinal cells at the transplantation site. The detectable rejection process shows that the posterior part of the eye is not absolutely immunologically privileged and that rejection is a serious problem in human retinal transplantation. Many questions concerning transplantation technique, graft treatment and postoperative treatment will be answered through more intensive experiments and clinical trials regarding the immunology. However, rejection of transplanted material is one of the main reasons why retinal transplantation has not yet proved successful.     

Is nebulin truly a component of the thin filament?

Thin filaments were prepared from rabbit and beef skeletal muscle with three different procedures, both at high and low ionic strength. Nebulin was always found to be associated with the myosin fraction and was always absent from the thin filament fraction.     

Temperature-induced structural changes in the myosin thick filament of skinned rabbit psoas muscle

By using synchrotron radiation and an imaging plate for recording diffraction patterns, we have obtained high-resolution x-ray patterns from relaxed rabbit psoas muscle at temperatures ranging from 1 degree C to 30 degrees C. This allowed us to obtain intensity profiles of the first six myosin layer lines and apply a model-building approach for structural analysis. At temperatures 20 degrees C and higher, the layer lines are sharp with clearly defined maxima. Modeling based on the data obtained at 20 degrees C reveals that the average center of the cross-bridges is at 135 A from the center of the thick filament and both of the myosin heads appear to wrap around the backbone. At 10 degrees C and lower, the layer lines become very weak and diffuse scattering increases considerably. At 4 degrees C, the peak of the first layer line shifts toward the meridian from 0.0047 to 0.0038 A(-1) and decreases in intensity approximately by a factor of four compared to that at 20 degrees C, although the intensities of higher-order layer lines remain approximately 10-15% of the first layer line. Our modeling suggests that as the temperature is lowered from 20 degrees C to 4 degrees C the center of cross-bridges extends radially away from the center of the filament (135 A to 175 A). Furthermore, the fraction of helically ordered cross-bridges decreases at least by a factor of two, while the isotropic disorder (the temperature factor) remains approximately unchanged. Our results on the order/disordering effects of temperature are in general agreement with earlier results of Wray [Wray, J. 1987. Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8:62a (Abstr.)] and Lowy et al. (Lowy, J., D. Popp, and A. A. Stewart. 1991. X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60:812-824). and support Poulsen and Lowy's hypothesis of coexistence of ordered and disordered cross-bridge populations in muscle (Poulsen, F. R., and J. Lowy. 1983. Small angle scattering from myosin heads in relaxed and rigor frog skeletal muscle. Nature (Lond.). 303:146-152.). However, our results added new insights into the disordered population. Present modeling together with data analysis (Xu, S., S. Malinchik, Th. Kraft, B. Brenner, and L. C. Yu. 1997. X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys. J. 73:000-000) indicate that in a relaxed muscle, cross-bridges are distributed in three populations: those that are ordered on the thick filament helix and those that are disordered; and within the disordered population, some cross-bridges are detached and some are weakly attached to actin. One critical conclusion of the present study is that the apparent order <--> disorder transition as a function of temperature is not due to an increase/decrease in thermal motion (temperature factor) for the entire population, but a redistribution of cross-bridges among the three populations. Changing the temperature leads to a change in the fraction of cross-bridges located on the helix, while changing the ionic strength at a given temperature affects the disordered population leading to a change in the relative fraction of cross-bridges detached from and weakly attached to actin. Since the redistribution is reversible, we suggest that there is an equilibrium among the three populations of cross-bridges.     

Motile properties of vimentin intermediate filament networks in living cells

PURPOSE: To evaluate the clinical utility and morphologic accuracy of gadolinium-enhanced excretory magnetic resonance (MR) urography after low-dose diuretic injection and to correlate the results with those of conventional urography. MATERIALS AND METHODS: In 71 patients with urologic symptoms, excretory MR urography was performed after intravenous injection of 5-10 mg furosemide and, 30-60 seconds later, 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight. The MR urograms were interpreted by three radiologists, who were blinded to the clinical outcome, and subsequently compared with conventional urograms. RESULTS: Injection of furosemide before contrast material led to rapid, uniform gadolinium distribution inside a sufficiently distended collecting system such that there was no excessive concentration of gadolinium in the urine. In patients with normal or moderately reduced excretory function, this effect allowed complete visualization of the urinary tract within 5-20 minutes of contrast material injection while minimizing gadolinium-related endoluminal T2* effects. The clinical course helped verify almost all MR urographic results. The MR urographic technique was significantly superior to conventional urography in the assessment of the ureters and bladder (P < .0001). Delineation of small caliceal abnormalities is still problematic. The best depiction of the pelvicaliceal system was obtained with fat-suppressed MR imaging, although it was still slightly inferior to conventional urography (P < .05). CONCLUSION: Gadolinium-enhanced excretory MR urography performed after low-dose diuretic injection is a promising and accurate alternative to conventional excretory urography for imaging the morphology of the urinary tract.