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A differential staining method for adenohypophyseal cells.

A modification of azan staining (Heidenhain, 1915) for a better differential demonstration than previously of adenohypophyseal cells is reported. Human hypophyses were fixed in Bouin's solution, washed in 70% alcohol, dehydrated and then embedded in paraffin. Sections of about 5 microns thickness were cut and mounted on albumin-coated glass slides. These sections were gently oxidized with a mixture of potassium permanganate and sulphuric acid, and then stained according to the original azan technique; thereafter a procedure of staining with aniline blue was added. With this improvement, human adenohypophyseal cells were clearly classified into six groups on the basis of the color and intensity of staining of the cytoplasm. The alpha-acidophilic cells were stained clear red with azocarmine and the epsilon-acidophilic cells, orange-red with orange G, respectively. With aniline blue, beta-basophilic cells stained a deep blue, while delta-basophilic cells were more weakly stained. The gamma-chromophobic cells were faintly stained red, and the ACTH-cells very faintly blue. Collagenous fibers developed a blue color, while erythrocytes were orange-red to red. This modified method produces much better results in differential staining of the adenohypophyseal cells than either the original azan or Masson-Goldner staining methods.

Adult↗

Ontogeny of taste sense.

Developmental changes in the taste receptors and CNS, in physiological and behavioural patterns in fetus and postnatal animals constitute the ontogeny of taste. Tongue epithelial cells are modified into taste buds only with innervation and are seen to degenerate on denervation. On removal of a portion of the tongue, with its taste buds, the central projections of gustation also atrophy, indicating an interdependence of taste receptors and central areas for their development. Gradual transitions in electrical responses to taste stimuli are shown from fetal to adult stage. While responses to ammonium chloride are marked in early fetus, with advancements in age responses to a wide variety of chemicals are shown. Again, early in the development, fibers responsive to chloride are predominant while at a later stage fibers responsive to sodium increase in number. Behaviourally, human fetus and neonatal mammals showed increased swallowing movements on sweet taste and decreased movements on bitter taste which is somewhat at variance with electrophysiological evidence and reflects on the multimodal involvement of taste cell functional and behavioural dynamics with age.

Animals↗

[Cloning and expression analysis of a LIM-domain protein gene from cotton (Gossypium hirsuturm L.)].

LIM-domain protein plays an important role in various cellular processes, including construction of cytoskeleton, transcription control and signal transduction. Based on cotton fiber EST database and contig analysis, the coding region of a cotton LIM-domain protein gene (GhLIM1) was obtained by RT-PCR from 4DPA (day post anthesis) ovule with fiber. The cloned fragment of 848 bp contains an open reading frame of 570 bp, coding for a polypeptide of 189 amino acids. It was demonstrated that the deduced GhLIM1 protein was highly homologous to the LIM-domain protein of sunflower (Helianthus annuus), tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Two intact LIM-domains, with the conserved sequence of a double zinc-finger structure (C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-24-C-X2-H), were found in the GhLIM1 protein. RT-PCR and Northern blot analysis showed that GhLIM1 gene expressed in root, shoot tip, hypocotyls, bud, leaf, anther, ovule and fiber (4DPA, 12DPA, 18DPA). However it was preferentially expressed in the shoot tip, fiber and ovule. It was proposed that the express of GhLIM1 gene is related to cotton fiber development.

Amino Acid Sequence↗

[Structural characterization of tissue-specific galactan from flax fibers by 1H NMR and MALDI TOF mass spectrometry].

A high-molecular-mass polysaccharide galactan (M 2000 kDa) was isolated from flax at the stage of cell wall thickening of the bast fiber development. The polymer structure was studied by 1H NMR spectroscopy and MALDI TOF mass spectrometry. It is built up of Gal (59%), Rha (15%), GalA (23%), and Ara (3%) residues. The galactan backbone consists of successively alternating monomer disaccharide units (--> 4GalA1 --> 2Rha1 -->)n and is similar in its structure to the backbone of rhamnogalacturonan-1 (RG-I). Rhamnose residues bear in position 4 beta-(1 --> 4)-galactose side chains of various lengths with a polymerization degree of up to 28 or higher. A part of the side chains have branchings.

Carbohydrate Conformation↗

[Possibilities of early diagnosis of autonomic neuropathy in diabetics].

The possibilities of the diagnosis of autonomous neuropathy were investigated by the authors in 71 insulin-treated diabetes mellitus patients with the use of dynamic esophageal scintigraphy and determination of cardiac reflex tests. It has been found that the extent and severity of the lesion can be earlier and more exactly recognized with the help of these two methods. Important information can be obtained by the determination of esophageal transit time about the functional condition of vagal nerves supplying gastrointestinal organs. In patients suffering from autonomous neuropathy the esophageal transit time proved to be significantly prolonged (p less than 0.01). The injury of parasympathetic heart innervation is earlier indicated by the pulmo-cardiac reflexes, while the troubles of symphathic functions are shown more responsively by the sustained handgrip test. Cases with considerable degree of orthostatic hypotension were not observed, its incidence may be probably higher only in advanced forms of autonomous neuropathy. The injury of vegetative nerve fibers develops mostly parallel in diabetes mellitus, but partial functional disorders can be also observed. Detailed neurological examinations were performed in 30 patients, and it has been found, that the lesion of peripheral nerves can be only later observed. Specific complications involving the nervous system are more frequent in cases with longer duration of the disease. In the authors opinion, for investigation and follow-up of larger number of diabetic patients, the dynamic esophageal scintigraphy and determination of pulmo-cardiac reflex and sustained handgrip tests seem to be suitable.

Adult↗

[Effect of phosphate and acidosis on the calcium sensitivity of the cardiac myofibrils].

The treatment of the bundles of rat myocardial fibers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N-tetraacetate (EGTA) made the sarcolemma permeable for ions and small molecules. At the incubation medium pH 7.0 the EGTA-treated fibers developed a half-maximal tension at pCa 5.4, and the maximal tension at pCa 4.8. Inorganic phosphate (10 mM) reduced the maximal tension by 18 +/- 3% and decreased the calcium sensitivity of the myofibrils so that there was a shift of the pCa/tension curve by 0.3 unit to the right. Acidosis (pH 6.6) also decreased significantly the calcium sensitivity, while the presence of 10 mM phosphate produced additional depression of the calcium sensitivity. It is concluded that phosphate accumulation by the ischemic myocardium combined with acidosis may depress the contractility not only due to depletion of the free calcium concentration in the myoplasm but also as a result of the reduced calcium sensitivity of myofibrils.

Acidosis↗

Physiological properties of zebrafish embryonic red and white muscle fibers during early development.

The zebrafish is a model organism for studies of vertebrate muscle differentiation and development. However, an understanding of fish muscle physiology during this period is limited. We examined the membrane, contractile, electrical coupling, and synaptic properties of embryonic red (ER) and white (EW) muscle fibers in developing zebrafish from 1 to 5 days postfertilization. Resting membrane potentials were -73 mV in 1 day ER and -78 mV in 1 day EW muscle and depolarized 17 and 7 mV, respectively, by 5 days. Neither fiber type exhibited action potentials. Current-voltage relationships were linear in EW fibers and day 1 ER fibers but were outwardly rectifying in some ER fibers at 3 to 5 days. Both ER and EW fibers were contractile at all ages examined (1 to 5 days) and could follow trains of electrical stimulation of up to 30 Hz without fatiguing for up to 5 min. Synaptic activity consisting of miniature endplate potentials (mEPPs) was observed at the earliest ages examined (1.2-1. 4 days) in both ER and EW fibers. Synaptic activity increased in frequency, and mEPP amplitudes were larger by 5 days. Miniature EPP rise times and half-widths decreased in ER fibers by 5 days, while EW fiber mEPPs showed fast kinetics as early as 1.2-1.4 days. ER and EW muscle fibers showed extensive dye coupling but not heterologous (red-white) coupling. Dye coupling decreased by 3 days yet remained at 5 days. Somites were electrically coupling, and this allowed filtered synaptic potentials to spread from myotome to myotome. It is concluded that at early developmental stages the physiological properties of ER and EW muscle are similar but not identical and are optimized to the patterns of swimming observed at these stages.

Animals↗

Critical role of the capsaicin-sensitive nerve fibers in the development of the causalgic symptoms produced by transecting some but not all of the nerves innervating the rat tail.

We investigated the role of capsaicin-sensitive small diameter fibers in the development of the thermal and mechanical allodynia in a new rat model for neuropathic pain, produced by transecting some but not all of the nerves innervating the tail. Capsaicin (50 mg/kg, s.c.) injected neonatally prior to the nerve injury produced thermal hypoalgesia in the tail the degree of which was variable across individual rats, presumably as a result of variable degeneration of the small diameter fibers. When subjected to the nerve injury, the animals with moderate thermal hypoalgesia exhibited signs of pain (e.g., tail flick) to normally innocuous mechanical stimuli applied to the tail with von Frey hairs (4.9 mN or 19.6 mN bending force), but not to thermal stimuli given by immersion of the tail into cold (4 degrees C) or warm (40 degrees C) water. The animals with marked thermal hypoalgesia, on the other hand, exhibited no signs of pain either to the mechanical or to the thermal stimuli. These results suggest that the capsaicin-sensitive fibers are critical in the development of both the mechanical and thermal allodynia. It is hypothesized that the destruction of A delta- and C-nociceptive fibers by capsaicin prevented activities induced in these fibers by the nerve injury from producing a central sensitization and thus allodynia.

Animals↗

[Development of core/targetoid fibers in the diaphragm of chronically hypercapnic rats].

We tested the hypothesis that chronic hypercapnia contributes to the development of core/targetoid fibers in ventilatory muscles. Five Wistar rats were kept in 5% CO2 in air for 13 weeks. Arterial blood gases (mean +/- SE) were pH 7.402 +/- 0.017, PaCO2 47.4 +/- 2.4 Torr, and PaO2 105.2 +/- 13.2 Torr. While the rats were chronically hypercapnic, their mean minute ventilation was about 1.7 times higher than that of rats that breathed air. This level was maintained throughout the exposure to CO2. Approximately 6% to 12% of the diaphragmatic type-I fibers were replaced by core/targetoid fibers in 3 of 5 chronically hypercapnic rats but not in the 3 control rats. Chronic hypercapnia may have continuously stimulated the respiratory center and resulted in more work done by the ventilatory muscles. Long-term overuse of the ventilatory muscles, which may occur in patients with chronic respiratory failure, can cause morphological changes in type-I fibers in the diaphragm.

Animals↗

Muscle fiber pattern is independent of cell lineage in postnatal rodent development.

Muscle fibers specialized for fast or slow contraction are arrayed in characteristic patterns within developing limbs. Clones of myoblasts analyzed in vitro express fast and slow myosin isoforms typical of the muscle from which they derive. As a result, it has been suggested that distinct myoblast lineages generate and maintain muscle fiber pattern. We tested this hypothesis in vivo by using a retrovirus to label myoblasts genetically so that the fate of individual clones could be monitored. Both myoblast clones labeled in muscle in situ and clones labeled in tissue culture and then injected into various muscles contribute progeny to all fiber types encountered. Thus, extrinsic signals override the intrinsic commitment of myoblast nuclei to particular programs of gene expression. We conclude that in postnatal development, pattern is not dictated by myoblast lineage.

Animals↗

The effect of changing free Ca2+ on light diffraction intensity and correlation with tension development in skinned fibers of frog skeletal muscle.

The relationship between the diffraction intensity change of the first order line and tension development was examined in mechanically skinned single fibers from the dorsal head of the semitendinosus of frogs. Passive stretch of the fibers resulted in an increase in intensity over the range of sarcomere lengths from 2.5 to 3.6 microns, indicating that the intensity is a function of sarcomere length. Activation of skinned fibers caused a decrease in the intensity, at all sarcomere lengths, where the thick and thin filaments overlapped. The magnitude of the intensity decrease and that of the tension development depended on the Ca2+ concentration in the medium. The drop of intensity-pCa and the tension-pCa curves showed a similarly steep S-shape within a range of 0.5pCa unit, although the intensity-pCa curve shifted to the left; the pCa for 50% decrease in light signal was 6.48 and that for 50% tension development was 6.40. Caffeine (25 mM) added to the medium produced a decrease in the intensity of skinned fibers with the simultaneous development of tension, thereby indicating that caffeine induces a release of Ca2+ from the sarcoplasmic reticulum and disorder in the filaments ensues. Changes in diffraction intensity with electrical stimulation to the intact single fiber were similar, although a more striking summation was observed in the optical response, as compared to the tension development. These results suggest that tension development upon stimulation can be monitored by assessing the magnitude of diffraction intensity decrease in the first order line, except for some shift in the short fiber.

Animals↗

Formation of reticular fibers in the developing spleen of the chick embryo.

An electron microscope study was made to elucidate the manner in which reticular fibers form in the developing spleen of the chick embryo. On the 7th day of incubation, the splenic reticulum consisted of dendritic reticular cells without reticular fibers. From the 9th day onward, the intercellular gaps between processes of adjacent reticular cells were expanded, becoming occupied by fine flocculent materials. With the growth of the embryos, these materials were aggregated into felt-like bands, exhibiting the characteristics of mature reticular fibers. In the interstices of the reticulum, there appeared microfibrils of 8-30 nm in diameter. The reticular cells possessed numerous free ribosomes and polysomes, well-developed Golgi apparatuses, dilated rough-surfaced endoplasmic reticula, and many coated vesicles. The coated vesicles contained fine flocculent materials as presumable fiber precursors. The vesicles were enlarged as they approached the cell periphery, where they appeared to discharge their contents by reverse pinocytosis. some vesicles close to the cell membrane contained ruthenium red-positive materials which were considered to be glycosaminoglycan in nature and to compose the matrix of fibers. These materials also seemed to be secreted by reverse pinocytosis, being densely accumulated in the intercellular gaps. The differentiation of fibrous proteins into felt-like reticular fibers or striated microfibrils seems, therefore, to be intimately associated with the amount of glycosaminoglycan.

Animals↗

Development of oxytalan fibers in the rat molar periodontal ligament.

Although oxytalan fibers are known to be a ubiquitous component of the periodontal ligament, little information has been available concerning their organization in the developing periodontal ligament. In the present study, growth and distribution of oxytalan fibers were examined in the developing periodontal ligament of rat molars aged 11, 14, 19, 21 and 28 days. A quantitative analysis of the fibers was made and the spatial relationship between the fibers and blood vessels was studied by means of a three-dimensional reconstruction of serial sections. At the beginning of root formation, oxytalan fibers appeared at first as dot-like structures around the root sheath as well as in areas very close to blood vessels. These structures were resolved in the electron microscope to be made up of 12-nm-wide microfibrils in the vicinity of the surface of the cells of the root sheath. In the process of development, these dot-like structures elongated into entities with helical appearances. As the development further proceeded, longer oxytalan fibers were produced in the apico-occlusal direction along with blood vessels. Quantitative analysis showed that an increase in oxytalan fibers coincided with an increase in the density of the vascular network in the developing periodontal ligament. Based on the results of the present study, the role of oxytalan fibers in the developing periodontal ligament may be in the maintenance of the integrity of the vascular system as previously suggested.

Age Factors↗

Development of a fiber optic probe to measure NIR Raman spectra of cervical tissue in vivo.

The goal of this study was to develop a compact fiber optic probe to measure near infrared Raman spectra of human cervical tissue in vivo for the clinical diagnosis of cervical precancers. A Raman spectrometer and fiber optic probe were designed, constructed and tested. The probe was first tested using standards with known Raman spectra, and then the probe was used to acquire Raman spectra from normal and precancerous cervical tissue in vivo. Raman spectra of cervical tissue could be acquired in vivo in 90 s using incident powers comparable to the threshold limit values for laser exposure of the skin. Although some silica signal obscured tissue Raman bands below 900 cm-1, Raman features from cervical tissue could clearly be discerned with an acceptable signal-to-noise ratio above 900 cm-1. The success of the Raman probe described here indicates that near infrared Raman spectra can be measured in vivo from cervical tissues. Increasing the power of the excitation source could reduce the integration time to below 20 s.

Biopsy↗

Transient appearance of calcitonin gene-related peptide-like immunoreactive fibers in the developing cerebellum of the rat.

A plexus of calcitonin gene-related peptide (CGRP)-containing fibers were transiently found in the developing cerebellum of the rat by means of the indirect immunofluorescent method. CGRP-like immunoreactive fibers appeared in the cerebellum by embryonic day 22. Immunoreactive fibers rapidly increased and these made a dense plexus in the Purkinje cell layer by postnatal day 2. However, only a few if any immunoreactive fibers were seen in the Purkinje cell layer or molecular layer of adult rats.

Animals↗

Calcitonin gene-related peptide immunoreactive nerve fibers in the developing salivary glands of the rat.

The appearance and distribution of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibers was studied by indirect immunofluorescence microscopy in the developing salivary glands of the rat at daily age intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 16th, 30th postnatal (p.n.) days. The findings were compared to samples of adult rats. CGRP-IR fibers appeared on day 20 i.u. both in parotid and submandibular glands. In submandibular glands, fibers were first seen surrounding the developing ductal branches, in the mesenchyme and also in association with developing ducts and blood vessels. In the parotid gland CGRP-IR fibers first appeared around ductal branches and blood vessels on day 20 i.u. and on day 21 i.u. CGRP-IR nerve fibers were found in the mesenchyme and around developing ducts. The density of CGRP-IR fibers was highest between the 2nd and 16th postnatal days, after which the amount of CGRP immunoreactivity slowly decreased to the levels of adult glands. The increase in a number of CGRP-IR nerve fibers especially around the ductal and vascular elements in the developing salivary glands indicates that they may play a role in the functional maturation of salivary glands.

Animals↗

Effect of wheat bran fiber on the development of mammary tumors in female intact and ovariectomized rats treated with 7,12-dimethylbenz(a)anthracene and in mice with spontaneously developing mammary tumors.

We examined the effect of consumption of graded increases of dietary fiber (soft white wheat bran) on the development of mammary gland carcinomas in intact female Sprague-Dawley rats during the promotion stage of carcinogenesis, induced with 7,12-dimethylbenz(a)anthracene (DMBA). The percent of rats with mammary carcinomas, the total number of mammary carcinomas and the mean number of mammary carcinomas per rat were reduced significantly at all fiber levels examined compared to rats fed a control diet. Inclusion of 9.6% fiber in the diets of ovariectomized rats that had been treated with a single i.v. dose of 2.5 mg DMBA/100 g body weight 2 weeks prior to removal of the ovaries resulted in a significant decrease of carcinomatous and benign mammary tumors compared to ovariectomized rats fed a control diet. Development of spontaneous mammary carcinomas in virgin C3H/HeOuJ female mice and growth of a transplantable mammary gland tumor in such mice were reduced by inclusion of 9.6% fiber in the diet, a reduction that was significant or just barely missed significance, depending on the source of the fiber. Our observations provide evidence that inclusion of soft white wheat bran in the diet is effective in the suppression of mammary gland tumorigenesis in an array of experimental animal models.

9,10-Dimethyl-1,2-benzanthracene↗

Shortening velocity and myosin heavy chains of developing rabbit muscle fibers.

The regulation of vertebrate muscle contraction with respect to the role of the different subunits of myosin remains somewhat uncertain. One approach to gaining a better understanding of the molecular basis of contraction is to study developing muscle which undergoes changes in myosin isozyme composition and contractile properties during the normal course of maturation. The present study utilizes single fibers from psoas muscles of rabbits at several ages as a model system for fast-twitch muscle development. This approach eliminates the inherent problems of interpreting results from studies on whole muscles which usually contain heterogeneous fiber types with respect to contractile properties and isoenzyme composition. Maximum velocity of shortening and tension-generating ability of individual fibers were measured and the myosin heavy chain composition of the same fibers was examined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel system. The results indicate that 1) with regard to contractile properties, there is a transitional period from slow to fast shortening velocities within the first postnatal month; 2) a strong, positive correlation exists between the speed of shortening and tension-generating ability of individual postnatal day 7 fibers, suggesting that as more myosin is incorporated in these developing fibers it is of the fast type; and 3) there is a wide variation in maximum velocity of shortening among postnatal day 7 psoas fibers which is also a time when a mixture of heavy chain isoforms characterizes the myosin composition of single muscle fibers.

Aging↗