Ultrastructure and innervation of the swimbladder of Tetractenos glaber (Tetraodontidae)

Summary The general structure, ultrastructure and innervation of the swimbladder of the smooth toadfish, Tetractenos glaber, were examined with light-microscopic, fluorescence-histochemical, and transmission electron-microscopic techniques. The structure of the swimbladder is similar to that of other euphysoclists. Fluorescence histochemistry showed adrenergic fibres in both the secretory and resorptive areas of the swimbladder. Transmission electron microscopy revealed two morphologically distinct axon profiles type-I profiles containing many small, flattened vesicles; type-II profiles containing both large, granular vesicles and rounded, small clear vesicles in varying proportions.The gas-gland cells and surrounding muscularis mucosae are innervated by both type-I and type-II fibres. Type-I fibres also innervate pre-rete arteries. The rete- and gas-gland capillaries do not appear to be innervated. Arteries running to the resorptive area are innervated by type-I fibres. Both type-I and type-II profiles make contact with the muscularis mucosae in the resorptive area. Only type-I fibres innervate the radial dilator muscle in the oval sphincter region, whereas only type II fibres innervate the circular muscle of the oval sphincter.Type-I fibres took up -methyl-noradrenaline, and could not be found after pre-treatment with 6-hydroxydopamine. They are, therefore, assumed to be adrenergic. Type-II fibres were tentatively identified, by exclusion, as cholinergic.     

Ultrastructural and immunocytochemical studies of neuromuscular junctions in oviduct of Locusta migratoria

The ultrastructure of nerve endings in the oviduct visceral muscles of Locusta migratoria was studied by electron microscopy and by immunogold labeling for two kinds of neuromodulators, the pentapeptide proctolin and FMRFamide-related peptides. Nerve endings contained electron-lucent round vesicles and two kinds of granules (round and avoid), and formed two types of synapses or release sites with the muscle. The morphologically distinct nerve endings were classified into three different categories based on the composition of synaptic vesicles and granules. Type-I nerve endings were dominated by electron-lucent round vesicles and contained only a few round electron-dense granules. Type-II nerve endings contained mostly electron-dense round granules and electron-lucent round vesicles. A few electron-dense ovoid granules were also present. Electron-dense ovoid granules dominated the type-III nerve endings, which usually contained less electron-lucent vesicles than either type-I or II nerve endings. Both proctolin and FMRFamide-like immunoreactivity was associated with electron-dense round granules. However, FMRFamide-like immunoreactivity was only found in the type-II nerve endings, while proctolin immunoreactivity was found within type-I nerve endings as well as in some type-II nerve endings. Immunological results therefore allow us to further divide type-II nerve endings into type-IIa (immunonegative for proctolin) and type-IIb (immunopositive for proctolin). Type-III nerve endings show no immunolabeling to either proctolin or FMRFamide.     

Computational study of EGFR inhibition: molecular dynamics studies on the active and inactive protein conformations

The structural diversity observed across protein kinases, resulting in subtly different active site cavities, is highly desirable in the pursuit of selective inhibitors, yet it can also be a hindrance from a structure-based design perspective. An important challenge in structure-based design is to better understand the dynamic nature of protein kinases and the underlying reasons for specific conformational preferences in the presence of different inhibitors. To investigate this issue, we performed molecular dynamics simulation on both the active and inactive wild type epidermal growth factor receptor (EGFR) protein with both type-I and type-II inhibitors. Our goal is to better understand the origin of the two distinct EGFR protein conformations, their dynamic differences, and their relative preference for Type-I inhibitors such as gefitinib and Type-II inhibitors such as lapatinib. We discuss the implications of protein dynamics from a structure-based design perspective.     

Spectroscopic investigations of Panulirus interruptus hemocyanin in the crystalline state

Single-crystal ultraviolet spectroscopy, X-ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa. Three types of crystals have been investigated. Type-I monoclinic crystals, which have been used for the X-ray structure determination, contain virtually only deoxyhemocyanin. Type-II monoclinic crystals, which are less well ordered than the type-I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type-II monoclinic form, subject to gradual conversion to methemocyanin.     

Glucan-phosphorylase forms in cotyledons of Pisum sativum L.: Localization,developmental change,in-vitro translation,and processing

The occurrence, location, and biosynthesis of glucan-phosphorylase (EC 2.4.1.1) isoenzymes were studied in cotyledons of developing or germinating seeds of Pisum sativum L. Type-I and type-II isoenzymes were detected, and were also localized by indirect immunofluorescence using polyclonal anti-type-I or anti-type-II phosphorylase antibodies. Type-I isoenzyme was found in the cytosol of parenchyma cells whereas the type-II enzyme form is a plastid protein which resides either in amyloplasts (in developing seeds) or in proplastids (in germinating seeds). During seed development, type-II phosphorylase was the predominant isoenzyme and the type-I isoenzyme represented a very minor compound. During germination, the latter increased whilst type-II phosphorylase remained at a constant level. In in-vitro translation experiments, type-I isoenzyme was observed as a final-size product with an apparent molecular weight of approx. 90 kDa. In contrast, type-II phosphorylase was translated as a high-molecular-weight precursor (116 kDa) which, when incubated with a stromal fraction of isolated intact pea chloroplasts, was processed to the size of the mature protein (105 kDa).Abbreviations IgG immunoglobulin G - kDa kilodalton - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft. The authors are endebted to Mrs. Karin Niehüser for help in the immunocytochemical studies.     

A low-voltage wide-input CMOS comparator for sensor application using back-gate technique

In this paper, two new analog CMOS comparators (Type-I and Type-II) with low-voltage and wide-input capabilities are proposed. The comparator receives two analog inputs and puts out one digital state to identify the larger (or the smaller) of the input variables, which represents an useful operation in data conversion and sensory signal processing. Without using special fabrication technologies, the supply voltage of the circuit is reduced to 1 V. Due to the utilization of CMOS back-gate technique, the input range of the comparators is greatly improved. The comparators are composed of bulk-driven stage and dynamic latch. By using a CMOS n-well technology, the results of HSPICE simulations indicate that the response time of Type-I circuit is 1 micros under 10 mV identified resolution. Type-II comparator achieves 5 mV identified resolution. The input dynamic ranges of the comparators are approximately rail-to-rail.     

Bile salt activation of cerebroside sulphate sulphohydrolase.

The cholate and taurodeoxycholate activations of cerebroside sulphate sulphohydrolase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) activity of arylsulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) were compared. Taurodeoxycholate caused a sharp peak of response at a concentration of 1.25 mg/ml (type-I activation). Cholate also showed type-I activation but, in addition, it evoked a second, higher, response plateau at concentrations between 5 and 10 mg/ml (type-II activation). At the pH of the reaction, cholate is converted largely to the sparingly soluble free aicd, so at the high concentrations associated with type-II activation, copious precipitates were formed. It was found that the precipitated material was essential for the type-II activation. Type-I activation appears to involve bile salt interaction with substrate, while type-II activation appears to involve enzyme interaction with solid-phase cholic acid. the putative mutant arylsulphatase A in an unusual form of metachromatic leukodystrophy hydolysed cerebroside sulphate only in the presence of high levels of cholate. The type-II activation may thus be simulating a physiological desulphation reaction.     

Diversity of the particulate methane monooxygenase gene in methanotrophic samples from different rice field soils in China and the Philippines

Methanotrophic bacteria play a crucial role in regulating the emission of CH4 from rice fields into the atmosphere. We investigated the CH4 oxidation activity together with the diversity of methanotrophic bacteria in ten rice field soils from different geographic locations. Upon incubation of aerated soil slurries under 7% CH4, rates of CH4 oxidation increased after a lag phase of 1-4 days and reached values of 3-10 micromol d(-1) g-dw(-1) soil. The methanotrophic community was assayed by retrieval of the pmoA gene which encodes the a subunit of the particulate methane monooxygenase. After extraction of DNA from actively CH4-oxidizing soil samples and PCR-amplification of the pmoA, the community was analyzed by Denaturant Gradient Gel Electrophoresis (DGGE) and Terminal Restriction Fragment Length Polymorphism (T-RFLP). DGGE bands were excised, the pmoA re-amplified, sequenced and the encoded amino acid sequence comparatively analyzed by phylogenetic treeing. The analyses allowed the detection of pmoA sequences related to the following methanotrophic genera: the type-I methanotrophs Methylobacter, Methylomicrobium, Methylococcus and Methylocaldum, and the type-II methanotrophs Methylocystis and Methylosinus. T-RFLP analysis detected a similar diversity, but type-II pmoA more frequently than DGGE. All soils but one contained type-II in addition to type-I methanotrophs. Type-I Methylomonas was not detected at all. Different combinations of methanotrophic genera were detected in the different soils. However, there was no obvious geographic pattern of the distribution of methanotrophs.     

Coping with aquatic hypoxia: how the plainfin midshipman (Porichthys notatus) tolerates the intertidal zone

Although plainfin midshipman (Porichthys notatus) are primarily known for their alternative reproductive tactics, and the dimorphic male subtypes, in which Type-I males demonstrate parental investment and mate attraction, and Type-II males ‘sneak’ fertilization and show no investment after fertilization, little is known about the physiology and tolerance to low aquatic oxygen while nesting in the intertidal zone. In May 2007, females and Type-I and Type-II males were collected, and in June 2009, only Type-I males were collected from nest sites on the coast of Vancouver Island, British Columbia. In the 2007 season, an initial assessment of hypoxia tolerance and nest parameters was recorded for the three subtypes of midshipman. Historical evidence indicates that Type-I males remain on the nest for prolonged periods, and our results suggest they can cope with repeated bouts of aquatic hypoxia by elevating their hematocrit and tolerating high lactate levels. The 2009 season was directed at examining the aquatic hypoxia tolerance of only the Type-I male. Hypoxic (~15 % air saturated water) Type-I males had oxygen consumption rates at ~12 % of the normoxic control (~100 % air saturated water) and a P_crit, the critical oxygen tension, when a fish switches from oxyregulator to oxyconformer, could not be determined; an indication that these fish are solely oxyconformers. With prolonged exposure to aquatic hypoxia, Type-I males displayed significant elevations in plasma and tissue lactate (heart), tissue glucose (liver), and a depression in gill Na⁺/K⁺ATPase and catalase activities. Results suggest that male Type-I midshipman survival in the intertidal zone is enhanced by metabolic depression and tolerance to anaerobic byproducts.     

The adenosine triphosphatase-inhibitor content of bovine heart submitochondrial particles. Influence of the inhibitor on adenosine triphosphate-dependent reactions.

1. The activity of the ATPase (adenosine triphosphatase) of phosphorylating particles prepared by sonication of bovine heart mitochondria in the presence of MgCl2 and ATP is influenced by the isolation method for the mitochondria used in the preparation of particles. Type-I particles, made from mitochondria isolated in a medium lacking succinate, have a lower ATPase activity than to Type-II particles, which are prepared from mitochondria isolated in a medium containing succinate. 2. Centrifugation under appropriate energized conditions increases the ATPase activity of Type-I particles almost to that of the Type-II particles. The ATPase activity of Type-II particles was only slightly stimulated by this procedure. These data are interpreted as indicating a higher content of the ATPase-inhibitor protein in the Type-I particles. 3. A comparison was made of the ATP-driven enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and the exchange of the endogenous tightly bound nucleotides of the ATPase in Type-I and Type-II particles. The effect of exogenous inhibitor protein on both these reactions was also studied. 4. The time-scale on which the inhibitor protein can exchange between ATPase molecules is discussed.     

Development and androgen regulation of the secretory cell types of the Syrian hamster (Mesocricetus auratus) Harderian gland

The secretory cell types of the hamster Harderian glands were studied in both male and female Syrian hamsters. As previously demonstrated, female hamsters showed a single secretory cell type (type I), while male hamsters displayed two secretory cell types (type I and type II). Type-II cells were observed after the first month of age correlating with the increase in testosterone levels. The administration of testosterone to adult female hamsters resulted in a marked increase in the percentage of type-II cells without a significant increase in the number of mitotic figures. Very low levels of serum testosterone were able to maintain the percentage of type-II cells. Castration of male hamsters produced a decrease in the percentage of type-II cells. This drop correlated with the reduction in serum testosterone levels. The chronic administration of a luteinizing hormone-releasing hormone agonist to male Syrian hamsters induced a significant reduction in both serum luteinizing hormone and testosterone. However, the percentage of type-II cells was similar to that of control hamsters suggesting that very low levels of circulating testosterone are able to maintain the percentage of type-II cells. In a final experiment male Syrian hamsters were treated with the antiadrogen cyproterone acetate. No changes were observed in the percentage of type-II cells, whereas serum luteinizing hormone and testosterone levels were significantly modified. We concluded that (1) type-II cells differentiate from type-I cells; (2) gonadal androgens are the major factor controlling this differentiation; and (3) the disappearance of type-II cells after androgen deprivation occurs through holocrine and apocrine mechanisms. The possible implication of 5-reductase in the regulation of secretory cell types in the Harderian glands of hamsters is discussed.     

Molecular discrimination of type-I over type-II methionyl aminopeptidases

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.     

Sexual dimorphisms in the vocal control system of a teleost fish: morphology of physiologically identified neurons

In one species of vocalizing (sonic) fish, the midshipman (Porichthys notatus), there are two classes of sexually mature males--Types I and II--distinguished by a number of traits including body size, gonad size, and reproductive tactic. The larger Type-I males (unlike Type-II males and females) build nests, guard eggs, and generate several types of vocalizations. Sound production by Type-I males is paralleled by a proportionate increase of 600% in their sonic muscle mass. The motor volley from ventral occipital roots innervating the sonic muscles establishes their contraction rate and, in turn, the fundamental frequency of emitted sounds. Electrical stimulation of the midbrain in every male and female elicited a rhythmic sonic discharge as recorded in the occipital roots; however, the fundamental frequency was slightly, but significantly, higher (20%) in Type-I males. Intracellular recording from identified motoneurons and presumed presynaptic "pacemaker" neurons showed their synaptic and action potentials had the same frequency as that of the nerve volley in every male and female. Reconstructions of physiologically identified motoneurons and pacemaker neurons following intracellular horseradish-peroxidase (HRP) filling showed their somata and dendrites to be 100-300% larger in Type-I males. These data unambiguously show that the size of a target muscle is correlated with the size of both the respective motoneurons and their presynaptic afferent neurons. As discussed, this implies that the dramatic increase in neuron size in the sonic motor system of Type-I males is causally dependent upon expansion of the sonic muscle. It is further likely that the more modest sex difference in the rhythmic central discharge is established by the intrinsic membrane properties of sonic neurons. These results also corroborate, at a number of behavioral, morphological, and neurophysiological levels, that the sonic motor system of "sneak spawning" Type-II males is similar to that of females. Thus, unlike the vocalizing Type-I males, sexual differentiation of the reproductive system in Type-II males is not linked to concomitant changes in the neurophysiological and morphological features of the sonic motor circuit.     

Type-II Myocardial Infarction – Patient Characteristics,Management and Outcomes

Background

Type-II MI is defined as myocardial infarction (MI) secondary to ischemia due to either increased oxygen demand or decreased supply. This categorization has been used for the last five years, yet, little is known about patient characteristics and clinical outcomes. In the current work we assessed the epidemiology, causes, management and outcomes of type II MI patients.

Methods

A comparative analysis was performed between patients with type-I and type-II MI who participated in two prospective national Acute Coronary Syndrome Israeli Surveys (ACSIS) performed in 2008 and 2010.

Results

The surveys included 2818 patients with acute MI of whom 127 (4.5%) had type-II MI. The main causes of type-II MI were anemia (31%), sepsis (24%), and arrhythmia (17%). Patients with type-II MI tended to be older (75.6±12 vs. 63.8±13, p<0.0001), female majority (43.3% vs. 22.3%, p<0.0001), had more frequently impaired functional level (45.7% vs. 17%, p<0.0001) and a higher GRACE risk score (150±32 vs. 110±35, p<0.0001). Patients with type-II MI were significantly less often referred for coronary interventions (36% vs. 89%, p<0.0001) and less frequently prescribed guideline-directed medical therapy. Mortality rates were substantially higher among patients with type-II MI both at thirty-day (13.6% vs. 4.9%, p<0.0001) and at one-year (23.9% vs. 8.6%, p<0.0001) follow-ups.

Conclusions

Patients with type-II compared to type-I MI have distinct demographics, increased prevalence of multiple comorbidities, a high-risk cardiovascular profile and an overall worse outcome. The complex medical condition of this cohort imposes a great therapeutic challenge and specific guidelines with recommended medical treatment and invasive strategies are warranted.     

Multinucleate nature,and mating by use of isozyme analysis inPoria cocos

Double staining study of nuclei and cell walls inPoria cocos indicated that the hyphal cells were multinucleate and had no clamp connections. Isozyme analysis of alcohol dehydrogenase (ADH) in 52 natural isolates revealed that there were three types of banding patterns: type I, five bands; type II, one slow band; type III, one fast band. Regenerants expressing type-II or type-III ADH-isozyme pattern were obtained from type-I isolates via protoplast manipulation. When the type-II regenerants were mated with the type-III regenerants, hyphae of type-I phenotype appeared. These data indicated that these type-II and type-III regenerants derived from protoplasts of the type-I isolates were primary hyphae. These primary hyphal cells were also multinucleate. Inter-strain mating ofP. cocos was performed and confirmed by ADH-isozyme analysis. Confronting cultures of a type-III regenerant derived from protoplasts of a type-I isolate and a type-II regenerant derived from a type-II isolate resulted in type-I hyphae.     

Phylogenetic Analysis of Different Breeds of Domestic Chickens in Selected Area of Peninsular Malaysia Inferred from Partial Cytochrome b Gene Information and RAPD Markers

The present investigation was carried out in an attempt to study the phylogenetic analysis of different breeds of domestic chickens in Peninsular Malaysia inferred from partial cytochrome b gene information and random amplified polymorphic DNA (RAPD) markers. Phylogenetic analysis using both neighbor-joining (NJ) and maximum parsimony (MP) methods produced three clusters that encompassed Type-I village chickens, the red jungle fowl subspecies and the Japanese Chunky broilers. The phylogenetic analysis also revealed that majority of the Malaysian commercial chickens were randomly assembled with the Type-II village chickens. In RAPD assay, phylogenetic analysis using neighbor-joining produced six clusters that were completely distinguished based on the locality of chickens. High levels of genetic variations were observed among the village chickens, the commercial broilers, and between the commercial broilers and layer chickens. In this study, it was found that Type-I village chickens could be distinguished from the commercial chickens and Type-II village chickens at the position of the 27th nucleotide of the 351 bp cytochrome b gene. This study also revealed that RAPD markers were unable to differentiate the type of chickens, but it showed the effectiveness of RAPD in evaluating the genetic variation and the genetic relationships between chicken lines and populations.