Microspectrophotometric Study Research Articles

Spectral absorbance changes in the photoreceptors of the juvenile masked greenling, Hexagrammos octogrammus (Pisces, Hexagrammidae).

When changing habitat during migration or ontogenesis, fish encounter changes of the visual environment, among which the most important is the spectral composition of light and turbidity. This leads to changes in the behavior, morphology, and physiology of the visual system, in particular, spectral sensitivity, which is determined by the properties of visual pigments. The masked greenling Hexagrammos octogrammus has an additional factor that shapes effective spectral sensitivity-the presence of densely bright orange cornea which reversibly changes its density depending on the state of light/dark adaptation. However, it is unknown to what extent the properties of visual pigments are matched to spectral properties of the developing cornea at the final stage of metamorphosis during migration of juveniles from pelagic to coastal environments. The findings of this microspectrophotometric study show that the range of the spectral sensitivity of rods and cones in juveniles is much wider than in adults, with a shift in the sensitivity of a significant part of the cells to shorter wavelengths. The reason for this is the large variation in the ratio of chromophores A1:A2 in the pigment mixture in each cell and possible expression of new opsins. This also indicates the asynchrony of pigment transformations in different types of photoreceptors and the incompleteness of these transformations on the eve of the transition to bottom life in shallow water.

UV–Vis microspectrophotometric study of wool and polyamide fibres dyed with analogous gryfalan dyes

The presented study was dedicated to the comparison between wool and polyamide fibres, dyed with metal-complex (gryfalan) dyes being analogous regarding type and concentration, with the use of UV–Vis microspectrophotometry. The affinity of used dyes to the particular fibre type as well as the influence of fibre polymer on the quality of spectra obtained were subjected to evaluation. The absorbance spectra, obtained with the use of a 20/20 PV™ microspectrophotometer (CRAIC Technologies) for single fibres selected from 15 woolen and 15 polyamide fabrics, were studied. The results indicated that at the same concentration of dye, the dyes' affinity is higher for polyamide fibres than for wool. Thus, the conduction of the examination of single wool fibres in the visible and in the UV-A range can be recommended, and in the case of analysing fibres with similar microscopic colour, the chemometric treatment seems to be an effective way of discrimination.

Aquatic Environments and Spectral Sensitivity of Rod Visual Pigments in the Retina of Rainbow Trout (Oncorhynchus mykiss): A Microspectrophotometric Study

The vertebrate retina is a part of the central nervous system (CNS) that can be visualized in a non-invasive manner. The retina is a thin multi-layered sensory tissue of neural cells that contains millions of photoreceptors. There are two physiologically distinct types of photoreceptors across the retina, the cones and the rods. The light sensitivity of the photoreceptors results from the presence of visual pigment molecules in the outer segment of the cone and rod cells. All visual pigments are composed of two components: retinal (vitamin A aldehyde), termed a chromophore, bound to a protein called opsin. The spectral absorption properties of a visual pigment are mainly determined by the amino acid sequence of the opsin protein and the type of chromophore (A1 or A2 or mixed) bound to the opsin. There is a shifting from A1- to A2-based chromophores in the same opsin due to different environmental factors, e.g. aquatic environments. A1/A2 visual pigment ratios play a dominant role in determining rod λmax in rainbow trout (Oncorhynchus mykiss), shifting the λmax of the visual pigment and spectral bandwidth of absorbance (HBW, half-band width). The spectral absorption characteristics of the retinal rod photoreceptors in the Rainbow Trout (Oncorhynchus mykiss) were measured using the charge-coupled device (CCD)- based microspectrophotometer (MSP) system. We investigated retina preparations of juvenile rainbow trout (Oncorhynchus mykiss) using 54 rod photoreceptors. The spectral sensitivity of each rod photoreceptor was calculated using λmax (wavelength of maximum absorbance) and HBW. The mean wavelength of maximum absorbance values for the alpha-bands were 503.5±2.5nm for rhodopsin- dominated rods, 523.1±7.9nm for porphyropsin-dominated rods, and 509.6±1.9nm for rhodopsin porphyropsin mixed rods. The half-band width (HBW) of the main absorption bands was 4567±771 cm-1 for vitamin A1-based visual pigments and 4697±487 cm-1 for vitamin A2-based visual pigments. Our results provide the evidence of shifting the λmax of the visual pigment to a longer wavelength, broadening spectral bandwidth of absorbance, and presence three fundamentally different classes of rod photoreceptors in the retina of the rainbow trout depending on A1/A2 visual pigment ratios due to different environmental variables, e.g. aquatic environments.

Wound effects in the xylem of poplar: A UV microspectrophotometric study

AbstractCell wall modifications in vessels and fibres of wound wood ofPopulus tremulaL.×P. tremuloidesMichx. formed after mechanical wounding have been examined by light microscopy, transmission electron microscopy, and UV microspectrophotometry (in scanning and point measurement mode), mainly focusing on the lignin distribution. With this goal, wound xylem within lateral wound callusis was collected after response periods of up to 23 months. Vessels and fibres in wound xylem deviated from their usual axial orientation. Vessels within the wound xylem were smaller in diameter and shorter in length. Xylem fibres were also shorter and developed thicker walls, especially in tissue adjacent to the wound. Cell walls and cell corners of these fibres showed on average a higher lignin content and a modified lignin composition. These wall changes probably enhance disease resistance of the wound tissue. With increasing distance from the wound edge, the modifications diminished and finally disappeared.

Role of melanin and artificial hair color in preventing photo‐oxidative damage to hair

This research is a multifaceted study which investigates not only the role of melanin in providing photostability to natural hair color and hair proteins, but also the claim that the presence of specific artificial colors in hair slows down the rate of photodegradation of hair proteins. In earlier studies, the extractability of protein from photodegraded hair was investigated and showed that many of the cleaved proteins could not be extracted because of photo‐oxidative cross‐linking. The current study investigates the effect of the amount of melanin in hair of different ethnicity and the presence of artificial hair colors on the extractability of the main classes of hair proteins. Furthermore, the data are used in the interpretation of the effect of these components in being able to prevent photo‐oxidative damage to hair proteins.When exposed to sunlight, hair undergoes changes in chemical, mechanical and morphological properties. The UVB and UVA regions of the solar spectrum are the most damaging to human hair. Of these two, the UVA region is predominant in the solar spectrum at low altitudes. Hair of different ethnicity responds differently to the damaging radiation of the solar spectrum, because of different amounts of melanin present in hair. Melanin absorbs the impinging radiation (especially at the lower wavelengths (254–350 nm), and converts it by some complex internal mechanism into heat. Because of this, melanin provides a photochemical protection to natural hair color and hair proteins and prevents their photodegradation. However, the melanin pigments act sacrificially and become themselves degraded in the process of protecting the proteins from light. As a result, this «protective» effect of the melanin pigments does not last during long‐term intense exposure, when, regardless of the amount of melanin in hair, most matrix, intermediate filament and high molecular weight hair proteins undergo photo‐oxidative cross‐linking into higher molecular weight species, and their extractability from hair decreases significantly.The goal of this study is to demonstrate how UV‐radiation affects natural and artificial hair color during long‐term exposures. Bright‐field and UV‐microspectrophotometry and an electrophoretic separation technique (SDS‐PAGE) were chosen as investigative techniques for these studies, because they are well‐suited to accurately and reproducibly investigate the initial properties of a specific hair sample and the changes in these properties as a result of long‐term light‐exposure. The goal of this paper is not to relate this to the content and type of melanin in hair. Electrophoresis, while not measuring the exact quantitative amount of protein extracted, is a semi‐quantitative method, where increases in brightness of the bands represent increased amounts of proteins that were extracted of that specific protein from hair. This electrophoretic study attempts to determine whether the presence of natural or artificial color in hair influences the protein extractability in unaltered hair and the photo‐oxidative cross‐linking during light‐exposure.The bright‐field microspectrophotometric study showed that high concentrations of melanin provide protection to the melanin itself and that they prevent loss of natural hair color during light‐exposure. However, neither large amounts of melanin in hair of different ethnicity, nor artificial hair colors (even a dye with an absorption in the UV region) provide protection to the hair proteins against photodegradation under the conditions used in this study.UV‐microspectrophotometry has suggested the formation of high levels of photo‐oxidized proteins as a result of light‐exposure. Electrophoresis revealed photo‐oxidative cross‐linking of most matrix, intermediate filament and high molecular weight hair proteins into their higher molecular weight analogues, rendering them less extractable due to their lowered diffusivity. Only very low levels of low molecular weight matrix proteins could be extracted.

Multiple photopigments from the Mexican blind cavefish, Astyanax fasciatus: a microspectrophotometric study

The cave-dwelling (hypogean) form of the teleost Astyanax fasciatus is blind, having only subdermal eye rudiments, but nevertheless maintains intact opsin genes. Second generation offspring of a cross between these and the normally sighted surface (epigean) form inherit opsin genes from both ancestries. A study of the expressed hypogean opsins of the hybrids, in comparison to the epigean forms, was undertaken by microspectrophotometry. The hybrid population showed considerable variation in the visual pigments of double cones, with evidence for two groups of cells with λ max intermediate to those of the epigean pigments. Possible explanations for these intermediate pigments are discussed, including the hypothesis that they may represent hybrid genes similar to the genes for anomalous cone pigments in humans. Evidence was also found for ultraviolet-sensitive single cones and for an additional MWS pigment.

Visual pigments and dichroism of anchovy cones: a model system for polarization detection.

The retinas of anchovies have two unique photoreceptor types: "bifid" and "long" cones (Fineran & Nicol, 1976). The outer segments of these cells contain multiple layers of membranes (lamellae) oriented longitudinally (axially). This orientation is distinct from that in all other vertebrate rods and cones, where the lamellae are stacked transversely with their planes perpendicular to the incident light path. Although the common arrangement provides optimal absorption for normally incident light rays, it is also insensitive to the rays' direction of vibration (i.e. their polarization). In contrast, the two mutually perpendicular sets of axially oriented lamellae segregated into bifid and long cones could function as the principal analyzers for linearly polarized light, as previously hypothesized (Fineran & Nicol, 1976, 1978). Here, we report on a microspectrophotometric study that shows (1) the presence of two spectrally distinct visual pigments in the three photoreceptor types of the bay anchovy retina; these are typical vertebrate pigments in that they bleach, when exposed to light, and have absorption spectra like all other vitamin A1-based visual pigments; (2) that the rods and cones exhibit dichroic absorption of light in accordance with their lamellar orientation, and (3) that the two cone types of the retina contain a spectrally indistinguishable pigment with peak absorbance (lambda(max)) around 540 nm, while the rods contain a rhodopsin-like pigment with lambda(max) near 500 nm. Compared to other vertebrates, anchovies are remarkable for using a monochromatic cone system with unusual specializations supportive of a polarization detection system.

Ionochromic properties of long-wave-sensitive cones in the goldfish retina: an electrophysiological and microspectrophotometric study

Long-wave-sensitive (LWS) cone visual pigments are sensitive to the concentration of chloride ions and show a spectral shift to shorter wavelengths when exposed to low chloride levels. We have used the aspartate-isolated mass receptor potential of the electroretinogram (ERG) to establish whether the spectrally shifted cone pigment is functional. In the goldfish, Carassius auratus, the λ max of the LWS porphyropsin is displaced from about 622 nm to around 606 nm when chloride is replaced by gluconate. The electrical response of the LWS cones (but not MWS cones and rods) is selectively and reversibly abolished by the removal of chloride ions. The effect is concentration dependent and a decrease to half the normal chloride ion concentration is sufficient to cause a decrease in the response.

An immunohistochemical study of cadherin 5 (VE-cadherin) in vascular endothelial cells in placentas with gestosis.

To evaluate vascular endothelial-cell functioning in placentas with gestosis, using the monoclonal antibody to cadherin 5. The extra-cellular moiety of cadherin 5 was transfected into L-cells to enable us to examine their cell-adhesion activity. The expression of cadherin 5 was evaluated in human umbilical-vein endothelial cells and in placentas with gestosis by immunostaining using the anti-cadherin 5 antibody. A microspectrophotometric study also was conducted of the placentas with gestosis. We determined the total base sequence for cadherin 5 and found that it is homologous with a known cadherin but is a new, unique clone. Cadherin 5 has cell-adhesion activity and is expressed in endothelial cells at the cell-adhesion surface. The expression of cadherin 5 in endothelial cells took place in the placentas with gestosis, but to a lesser extent than in normal placental endothelial cells. The reduced expression of cadherin 5 in placentas with gestosis suggests that endothelial cell functioning is impaired in placentas with gestosis. Cadherin 5 in endothelial cells might influence placental functions and fetal development.

A microspectrophotometric study on the physicochemical properties of antipyrylazo III injected into rat myoballs for measuring free magnesium ion

The accurate measurement of the intracellular concentration of free magnesium ions ([Mg 2+] i) is essential for evaluating the role of Mg 2+ in cellular functions. Among the specific (compared to fluorescent indicators) metallochromic dyes, antipyrylazo III (APIII) appears to be most suitable for measuring such concentrations in vertebrate cells according to in vitro studies. In this work, the intracellular physicochemical properties of APIII as a Mg 2+ indicator were investigated in the cultured rat myoball by means of a microspectrophotometric technique, in order to obtain an accurate measurement of [Mg 2+] i. A set of intracellular APIII–Mg 2+ calibration spectra was established after permeabilization of the cell membrane with saponin. In comparison with recordings obtained in K + solutions, the APIII absorption spectra recorded on a myoball exhibited a red shift and an overall change in absorbance, similar to that observed in a protein (bovine serum albumin: BSA) solution. The apparent dissociation constant of APIII for Mg 2+ in the myoplasm was found to be 3.16 mM, significantly higher than the 1.86 mM measured in K + solutions at the same pH (7.35). A stoichiometric ratio of 1:1 was found, however, both in solution and in the myoplasm. In addition, the injected APIII significantly affected the [Mg 2+] i of myoballs. The [Mg 2+] i was found to be 0.9±0.18 mM in 85 myoballs, on the basis of the intracellular calibration spectra obtained at the same myoplasmic APIII concentration (∼2.5 mM), and after correction for the perturbing effect of the dye. It is concluded that an intracellular calibration and recording of whole spectrum are essential for proper interpretations of intracellular dye signals.

A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas

The eyespot apparatus of the unicellular alga Chlamydomonas exhibits a clear directivity, i.e., it perceives light from different directions with different sensitivity. Using a newly constructed confocal microscope we have studied how absorption and reflection of eyespot and cell body shape this directivity. In agreement with previous results the eyespot was found to be highly reflectant, owing to its interference reflector design, but only for yellow light. Light of 490 nm, the maximum of absorption of the photoreceptor, was hardly reflected at all, even when the reflector was "tuned" to lower wavelengths by tilting it relative to the incoming light. The absorption of the carotenoids in the interference reflector also contributed little to the shielding properties of the cell, leaving the major contribution to the cell body. Thus most of the attenuation of light reaching the eyespot from the rear is due to chlorophyll and other pigments within the cell. In its peak around 490 nm the "contrast-ratio" reached a value of 8-10.

Cell cycle analysis in a multinucleate green alga, Boergesenia forbesii (Siphonocladales, Chlorophyta)*

SUMMARYThe cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1‐4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G1 period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G2 period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G1 stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G2 and mitosis. In other words, the cell cycle regulation of entrance into S phase from G1 is an important factor in the growth and morphogenesis in B. forbesii.

Role of DNA ploidy patterns in esophageal squamous cell carcinoma: An ultraviolet microspectrophotometric study

Controversy still exists about the influence of DNA content on the prognosis for patients with esophageal squamous cell carcinoma. DNA ploidy was determined by microspectrophotometric (MSPM) analysis of paraffin embedded malignant tissue from 78 patients with squamous cell carcinoma of the esophagus. The DNA distribution pattern was classified as diploid, low grade aneuploid (LGA), and high grade aneuploid (HGA) pattern. The relationships among DNA distribution patterns, pathologic features, clinical findings, and prognoses were investigated. Twenty-seven of 78 patients were also selected for analyses of the DNA ploidy pattern of lymph node metastases. Of 78 cancers, 20 (26%) were diploid, 15 (19%) LGA, and 43 (55%) HGA. The advanced carcinomas had the higher distribution of HGA pattern (55%), whereas the diploid pattern was more frequent in early stage (70%) or superficial (65%) esophageal cancer. In patients with the HGA pattern, there was a significantly higher frequency of lymph node metastases (79%) and marked lymphatic (74.4%) and vascular (69.8%) invasion compared with those exhibiting the LGA (33.3%) or diploid (25%) pattern. Five-year survival rates for patients with diploid carcinoma (57%) was significantly better than for those with the HGA pattern (14%). The distribution of nuclear DNA content was much narrower in the metastatic lymph node than in the corresponding primary cases. The HGA of DNA pattern based on spectrophotometry closely correlated with the factors generally indicative of the aggressive behavior of malignant tumors. Therefore, the role of the DNA ploidy pattern as a prognostic factor was emphasized.

A micro-spectrophotometric study on the spectral properties of phenol red injected into cytoplasm for pH i determination

The spectral properties of phenol red, used to determine intracellular pH (pH i), have been studied in the myoplasm of cultured rat skeletal muscle cells, by means of a micro-spectrophotometric technique. Two sets of intracellular calibration spectra at different dye concentrations were established in the presence of nigericin. Intracellular spectra of phenol red differ from the absorption spectra obtained in buffered solutions in the following ways: (1) Considerably greater absorbance in the violet region, owing to scattering of light by intracellular particles, which can be corrected by applying an energy transfer model derived from the work of Kubelka and Munk ( Z Tech. Phys. 12 (1931) 593). (2) Slight spectral red-shift and an increase in the dye p K d, assumed to be due to interaction of the dye with intracellular constituents, especially soluble proteins, since the same spectral modifications are observed in buffered solutions containing bovine serum albumin. The potential advantages of using phenol red to determine pH i are the simple dye-H + interaction, both in buffered solutions and in myoplasm, and the absence of dye selfassociations at the high dye concentrations required to perform intracellular measurements. Finally, our results show that absolute pH i can be measured to an accuracy of ±0.02 pH units using phenol red, if complete absorption spectra and intracellular spectral calibrations are available.

Nuclear DNA content of somatic and zygotic embryos ofVitis vinifera cv. Grenache noir at the torpedo stage

A flow cytometric analysis and an in situ DNA microspectrophotometric study were made concomitantly to establish why somatic grapevine (Vitis viniferacv. Grenache noir) embryos showed a low level of conversion into plantlets. In somatic embryos at the torpedo stage and in zygotic embryos at the same stage of development, ploidy level, DNA content per 2 C nucleus, and the cell-cycle state of the shoot apical meristem were examined. The frequency distribution histograms of nuclear DNA values were similar in the two types of embryos. At the torpedo stage both types of embryos had a majority of nuclei with 2 C DNA content equal to 1.6pg. In the shoot apices of somatic and zygotic embryos, DNA microspectrophotometry showed preferential blockage of the cell cycle at the G0–1 stage; however, 20% of somatic embryo shoot apices were blocked at the G0–2 stage. Analogies between somatic embryos and their zygotic homologues were shown. The genetical and environmental causes of the low level of conversion of grapevine somatic embryos into plantlets are discussed. Our work suggests that the in vitro culture conditions which were used could be incompatible with normal morphogenesis from the torpedo stage.

Visual pigments and oil droplets in genetically manipulated and carotenoid deprived quail: A microspectrophotometric study

The spectral absorbantes of visual pigments and retinal oil droplets were studied in three groups of Japanese quail ( Coturnix coturnix japonica): an unselected control population and two artificially selected strains that exhibited different early approach preferences between blue and red stimuli. The oil droplets were examined with and without prior carotenoid deprivation. Four cone pigments and five oil droplet types were identified, resembling those in other avian species. Carotenoid deprivation eliminated all pigmentation detectable in oil droplets by microspectrophotometry. Placement of chicks on normal diet gradually reintroduced normal pigmentation within the span of about a week. No statistically significant differences were found between normal and genetically selected birds in either visual pigments or oil droplet types, or in their relative proportions. It is concluded that differences in the early colour preferences of quail are unlikely to be a result of variation in the spectral properties of their photoreceptors.

DNA ploidy is associated with growth potential in gastric carcinoma

To correlate growth potential with DNA ploidy 109 patients with early gastric carcinoma and 132 patients with advanced gastric carcinoma were studied. Early gastric carcinomas were classified by growth potential into the small mucosal type, the superficially spreading (super) type, or the expansively penetrating (Pen A)/infiltratively penetrating (Pen B) types. Advanced gastric carcinomas were classified into funnel, column, or mountain types, each of which was divided further into expanding and infiltrative types. Cell nuclear DNA content was measured by microspectrophotometric study and classified as either low or high ploidy according to the degree of dispersion on the DNA histogram. Super type early and funnel type advanced carcinomas, characterized by superficially spreading growth, were more likely to have low DNA ploidy. In contrast, Pen A type early and column-expanding type advanced carcinomas, characterized by expansively penetrating growth, were more likely to have high DNA ploidy.

A microspectrophotometric study of DNA ploidy patterns of colorectal adenomas

Eighty-four adenomas (31 mild, 25 moderate, 28 severe atypia), 32 invasive carcinomas and 15 samples of normal mucosa were collected form polypectomy and surgically resected specimens, and their DNA ploidy patterns were examined by microspectrophotometry. The frequency of polyploidy and aneuploidy were 7% and 0% in normal mucosa, 13% and 0% in mild atypia, 20% and 44% in moderate atypia, 32% and 22% in severe atypia and 31% and 53% in invasive carcinomas, respectively. Furthermore, these abnormal ploidy patterns were found frequently in moderate atypia accompanying severe atypia and mild atypia accompanying moderate atypia. These findings suggest that adenomas with moderate atypia might be the early stage of histologically overt cancer. The thin-section method (4 microns or 7 microns) was useful for measuring regional DNA ploidy patterns of colorectal adenoma.

Effects of freezing-thawing on the spermatozoon nucleus: a comparative chromatin cytophotometric study in the porcine and human species

Freezing-thawing effects on the nuclei of porcine and human spermatozoa were studied by determining native DNA percentage from fluorescence after acridine orange (AO) staining and by analyzing chromatin structure by a quantitative microspectrophotometric study of Feulgen-DNA complexes before and after freezing. The study of boar spermatozoa revealed no alteration in native DNA percentage after freezing. However, native DNA percentage decreased significantly in human spermatozoa. Feulgen-DNA content and sperm nuclear surface area decreased in both species after freezing. These results prompted us to hypothesize an overcondensation of sperm chromatin after freezing-thawing. This overcondensation may be related to the lower conception rates obtained with human and porcine semen after cryostorage via defective decondensation.

Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs

Abstract Hyaluronate-mediated expansion of the extracellular matrix has been suggested as an important element of growth and morphogenesis in several developing systems. In vitro, various growth factors have been shown to stimulate hyaluronate synthesis as well as cell proliferation. A similar link between proliferation and hyaluronate production during in vivo growth is difficult to demonstrate, because in most systems the source of growth-promoting factors is either not known or not amenable to experimental manipulation. During amphibian limb regeneration, cell proliferation depends upon paracrine release of factors from axons in the limb stump, and the nerve supply can be eliminated or augmented experimentally for study of growth in this system. Denervated and amputated limbs of larval salamanders do not begin to regenerate until distal areas of the limb stumps are reinnervated. We have used such limbs to examine the effect exerted by the reappearance of nerves on the amount of hyaluronate in the tissue undergoing the growth response. Hyaluronate was demonstrated by the metachromatic dye Ethyl Stains-all, which stains hyaluronate blue while sulfated glycosaminoglycans (GAGs) and proteins in the extracellular matrix stain various shades of violet, and by microspectrophotometry of alcian-blue-stained GAGs in serial sections pretreated with buffer or with Streptomyces hyaluronidase (SH) to remove hyaluronate specifically. Both methods showed little hyaluronate in the distal region of limb stumps prior to reinnervation, while reinnervated stumps had amounts of hyaluronate similar to those of control blastemas. Autoradiography of 3H-glucosamine-labeled limbs indicated that hyaluronate in the blastemas of reinnervated limb stumps included material newly synthesized by cells throughout the growing tissue. The microspectrophotometric study revealed that the relative concentration of hyaluronate increased during the time distal limb areas were undergoing reinnervation, which was monitored by staining of nerve fibers. The increase in hyaluronate concentration was followed immediately by an increase in mitotic activity and a decrease in mesenchymal cell density, two changes leading to blastema formation that others have shown to be associated with reinnervation in this system. These observations indicate that the growth-promoting influence of nerves includes stimulation of hyaluronate production, an effect similar to that of serum or purified mitogens on many cultured cells. Hyaluronate synthesis appears to promote expansion of the limb stump, which occurs when denervated-amputated larval limbs are reinnervated.