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Nos últimos anos temos atendido vítimas de trauma com ferimento transfixante dos rins direito e esquerdo. Em trabalho anterior utilizamos colágeno tipo I em ferimentos transfixantes do fígado, com bons resultados. Assim, extendemos a utilização do método para o rim aplicando o colágeno tipo I produzido na UNICAMP, o qual pode ser injetado e moldado ocupando toda a área lesada e o trajeto anfractuoso ou irregular. Desta forma pode-se conseguir um tamponamento completo, coibindo a hemorragia e sustando a perda de urina. O colágeno será posteriormente reabsorvido não funcionando como corpo estranho. Trinta e seis cães foram submetidos a lesão transfixante na região inferior do rim esquerdo, procurando simular uma lesão produzida por um projétil de arma de fogo. Seis destes animais não receberam tratamento e foram a óbito por choque hemorrágico. Em todos os outros 30 animais, aplicou-se o colágeno tipo I em toda a extensão do túnel formado pela lesão, através da injeção do material. Foram realizadas avaliações clínicas no pós-operatório, sendo os cães sacrificados, em grupos de seis, aos 4, 7,15, 30 e 60 dias de pós-operatório O estudo macroscópico e microscópico, revelou uma formação cicatricial homogênea, com regeneração renal intensa, precoce e organizada, a partir do 7º dia de pós-operatório. Conclui-se que a aplicação do colágeno tipo I, nestes tipos de ferimentos são de aplicação simples e segura. Over the past years we have used of the type I collagen to tamponade transfixing hepatic lesions in dogs, manufactured at University of Campinas. We have considered the use of this collagen to tamponed transfixing renal lesions, wich can be molded to the entire wounded area thus tamponading all surface irregularities. In this way, a complete tamponade way be achieved, and urinary loss is prevented. Thirty six mongrel dogs received a perforating transfixing lesion in the distal region of the kidney, similar to that caused by a firearm bullet. Into the wounded tunnel type I collagen was introduced. Evaluations were made in the 4th , 7th, 15th, 30th and 60th day after the intervention, when animals were sacrificed. Microscope evaluation induced a homogeneous scar formation with early, organized renal regeneration evident from the 7th day onward. It is therefore believed that the use of type I collagen is an effective and safe method to be used in this type of renal wound.

Some basic topics concerned with the extraction of textural and geometric information from cell nucleus images as well as description and characterization of chromatin supraorganization and consequent classification of nuclear phenotypes are presented. Alguns tópicos básicos relativos à extração de informações texturais e geométricas de núcleos celulares, bem como a descrição e a categorização da supraorganização cromatínica e conseqüente classificação de fenótipos nucleares, são aqui apresentados.

BACKGROUND: The infrared (IR) analysis of dried samples of DNA and DNA-polypeptide complexes is still scarce. Here we have studied the FT-IR profiles of these components to further the understanding of the FT-IR signatures of chromatin and cell nuclei. METHODOLOGY/PRINCIPAL FINDINGS: Calf thymus and salmon testis DNA, and complexes of histone H1, protamine, poly-L-lysine and poly-L-arginine (histone-mimic macromolecules) with DNA were analyzed in an IR microspectroscope equipped with an attenuated total reflection diamond objective and Grams software. Conditions including polypeptides bound to the DNA, DNA base composition, and single-stranded form were found to differently affect the vibrational characteristics of the chemical groups (especially, PO(2)(-)) in the nucleic acid. The antisymmetric stretching (ν(as)) of the DNA PO(2)(-) was greater than the symmetric stretching (ν(s)) of these groups and increased in the polypeptide-DNA complexes. A shift of the ν(as) of the DNA PO(2)(-) to a lower frequency and an increased intensity of this vibration were induced especially by lysine-rich histones. Lysine richness additionally contributed to an increase in the vibrational stretching of the amide I group. Even in simple molecules such as inorganic phosphates, the vibrational characteristics of the phosphate anions were differently affected by different cations. As a result of the optimization of the DNA conformation by binding to arginine-rich polypeptides, enhancements of the vibrational characteristics in the FT-IR fingerprint could be detected. Although different profiles were obtained for the DNA with different base compositions, this situation was no longer verified in the polypeptide-DNA complexes and most likely in isolated chromatin or cell nuclei. However, the ν(as) PO(2)(-)/ν(s) PO(2)(-) ratio could discriminate DNA with different base compositions and DNA in a single-stranded form. CONCLUSIONS/SIGNIFICANCE: FT-IR spectral profiles are a valuable tool for establishing the vibrational characteristics of individualized chromatin components, such as DNA and DNA-polypeptide complexes in dried samples.

We use the optical path difference (OPD) technique to quantify the organization of collagen fibers during skin repair of full-thickness burns following low-intensity polarized laser therapy with two different polarization incidence vectors. Three burns are cryogenerated on the back of rats. Lesion L(parallel) is irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction. Lesion L(perpendicular) is irradiated using the electric field vector of the polarized laser radiation aligned perpendicularly to the aforementioned orientation. Lesion C is untreated. A healthy area labeled H is also evaluated. The tissue samples are collected and processed for polarized light microscopy. The overall finding is that the OPD for collagen fibers depends on the electric field vector of the incident polarized laser radiation. No significant differences in OPDs are observed between L(parallel) and H in the center, sides, and edges of the lesion. Lesions irradiated using the electric field vector of the polarized laser radiation aligned in parallel with the rat's occipital-caudal direction show higher birefringence, indicating that collagen bundles in these lesions are more organized.

The immortalized human breast epithelial cell line MCF-10F is an important tool for studies on experimental tumorigenesis induced by drugs, transfected Ha-ras oncogene, and hormones. Considering that many relevant data have thus far been established only for MCF-10F cells cultivated on glass, and that there are data showing different cell death ratios for tumorigenic cells obtained from benzo[a]pyrene (BP)-transformed MCF-10F cells cultivated on plastic compared with glass, nuclear parameters estimated by image analysis and cell death ratios were compared for cells grown on plastic and glass substrates differing in chamber surface sizes and working culture medium volumes. It was concluded that for slides with a growth size equal to 9.4 cm2, plastic substrate was more advantageous than glass for growing MCF-10F cells because although the apoptotic ratios (AR) for the cells grown on plastic are low as it would be expected for nontransformed cells, they are bigger than those reported for the BP-transformed MCF-10F cells cultivated on the same substrate but closer to those of the BP-transformed MCF-10F cells receiving a normal chromosome 17. In addition, the plastic substrate did not induce variable nuclear image results as those found in the latter. The 0.5-cm2-sized chambers on plastic slides proved to be inadequate for cell nuclear image analysis and cell death studies on account of the variable geometric, densitometric, and textural results and ARs produced and the unpublished consideration of a very slow growth rate generated under this growth condition.

Violacein, a pigment produced by Chromobacterium violaceum, is reported to be a potential drug for the treatment of Chagas' disease. Violacein is also effective against leukemia and lymphoma cells in culture (IC50 10−8 M). Changes in the nuclear acid content, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction and neutral red uptake in these cells were used to evaluate the cytotoxicity of violacein in V79 Chinese hamster (M-8) fibroblasts. Violacein was highly cytotoxic to V79 fibroblasts (IC50 5–12 μM). Using the TUNEL method and the Feulgen reaction coupled to image analysis, violacein (5 and 10 μM) was found to trigger apoptosis but not necrosis in V79 cells. The morphological changes seen in the nuclei of these cells included chromatin condensation and a decrease in deoxyribonucleic acid content. These results demonstrating that violacein induces apoptosis in V79 cells strengthen its potential as a therapeutic agent.

For the purpose of determining the changes in tensile strength and levels of macromolecular orientation in collagen bundles as a result of mechanical stimuli, rats were submitted to different experimental treatments such as physical exercise and muscular denervation. After these treatments the Achilles tendons of the animals were submitted to biomechanical and biophysical testing. An analysis of the "stress"-strain curves indicated that the tendons of trained animals possessed superior tensile strength characteristics as compared to their controls. The form birefringence curves (FBC) of the collagen bundles in these tendons also showed an increase in their state of aggregation of the extracellular matrix elements, while maintaining their levels of crystallinity. Average values of tensile strength were even higher in the tendons of denervated animals. An accentuation of the profile of the FBC was also observed in these tendons. The determination of the biomechanical and biophysical modifications can contribute to the understanding of the probable control mechanisms of the aggregation state patterns of the fibrous elements of the extracellular matrix.

With the objective of determining the apparent isoelectric point (pI) of the protein bodies of Glycine max in situ both before and after germination, seeds imbibed for 0 and 3 days were stained with methylene blue (MB) or xylene cyanol (XC) at various pH values (from 2.2 to 7.8), followed by cytophotometric measurements at λ=600 nm (MB) and λ=630 nm (XC). The results showed similar values for the protein material of cotyledon cells far from the vessels (pI=4.4±1), a progressive decrease in pI of the proteins located within the embryo after germination and pI=4.2±1 in the radicular parenchymatous cells, suggesting that for the mobilization of the reserve material there is an increase in the acidic groups of the protein bodies, which return to their initial quantity on reaching the interior of the growing cells.

Spectral absorption profiles at the UV and visible ranges were detected spectrophotometrically for solutions of various basic fuchsins, for the SCHIFF reagent prepared from these stains and for the end product from SCHIFF-formaldehyde reaction. The aim was to detect whether absorption spectra could vary as a function of the basic fuchsin origin up to the point of being suspected of affecting potentially the spectrum of FEULGEN-stained chromatin. The results indicate a constancy in the spectral absorption characteristics for the absorption profiles at the visible range, when one considers SCHIFF reagents prepared from various basic fuchsins added with aldehyde. However, it is possible that size, stereoarrangement and electron distribution of the aldehyde- (eventually, ketone-) bearing molecule involved in SCHIFF-carbonyl reactions would function as auxochroms, the spectral characteristics of which could be affected by dye content and contaminants of the basic fuchsin.

The autofluorescence of the Malpighian tubules of blood-sucking insects was studied microspectrofluorimetrically. Spectral emission profiles were determined for the naturally fluorescent substances present at the lumen of the proximal region and in the cytoplasm of the distal region of the organ. These were compared with the emission profiles exhibited by standards of fluorescent substances generally found in insect Malpighian tubules. The fluorescence detected at the proximal region was then concluded to be provided by uric acid whereas that of the distal region was attributed to kynurenine. The results did not vary as a function of the developmental stage of the insects. If pteridins are present in the tubules, they appear not to affect the fluorescence patterns determined in this work.

Critical electrolyte concentration (CEC) values were determined for DNA and DNA/Hl and DNA/protamine complexes under varying toluidine blue staining conditions and using Mg2+ as inorganic cation. CEC values were considered those molarities of MgCl2 at which green staining was detected microspectrophotometrically. Variation in the CEC values was found with respect to association of nuclear proteins with DNA and the nucleoprotein complex considered. The DNA filaments exhibited the highest range of CEC values, whereas the lowest CEC values were presented by the DNA/protamine complex, especially when the staining solutions were prepared in McIlvaine buffer. The method thus appears to be promising for further studies on differences among DNA/protein complexes in vitro or even in situ.

The pleochroism found in the histological sections dyed with toluidine blue buffered at pH 4 is caused by the selective absorption of polarized light by the molecule of the dye. This fact makes it possible to calculate the position of the molecules of acid mucopolysaccharides (AMP) by indirect method.

We stressed in our wourk the necessity of knowing the details of the interaction between the acid mucopolysaccharides and the collagen. Employing the appropriate methods we carried out the measurements of birefringence of strictly limited parts of collagen bundles. The measurements were carried out before and after the collagen bundles were subjected to the action of testicular hyaluronidase.

In this work, optical retardation (OR) was investigated in corneal stroma collagen fibers from nonobese diabetic (NOD) and healthy (BALB/c) mice. Linear dichroism (LD) was investigated in corneas stained with Ponceau SS and Toluidine Blue, using microspectrophotometer. NOD corneas showed higher OR values than the control. Ponceau SS-complexed collagen fibers showed positive LD that was higher for NOD corneas. After staining with Toluidine Blue, NOD and BALB/c corneas showed negative LD. The results of this study demonstrate that diabetes was capable of altering the corneal optical anisotropies, possibly altering the number of intermolecular crosslinks in collagen fibers.

Aims After undergoing lesions, tendons have disorganized collagen fibers compared to undamaged tendons. Arrabidaea chica leaves have the aglycones carajurin and carajurone, components of the antocyanins, with a strong pharmacological potential due to their healing properties. Thus, the aim of this study was to investigate the effect of topical application of A . chica extract during tendon healing. Main methods The calcaneal tendon of Wistar rats was partially transected with subsequent treatment with A . chica extract (2.13 g/mL) followed by excision on the 7th, 14th and 21st days. Control rats received only saline treatment. Key findings Transmission electron microscopy analysis showed the presence of a large amount of small segments of collagen fibrils in the transected region of the tendons on the 7th day in both the control and plant-treated groups. Considering the organization of the collagen fibers, higher values of birefringence were observed under polarization microscopy in the tendons of the plant-treated group on the 14th day compared to the control group. A larger quantity of dermatan sulfate was also detected after plant treatment in the same period. However, lesser dermatan and chondroitin sulfate were detected in the plant-treated group than in the control group on the 21st day. No differences were found in the values of birefringence between these groups. Intense metachromasy was observed in both transected groups on the 21st day. Significance In conclusion, the use of A . chica extract improves collagen organization and increases the quantity of dermatan sulfate on the 14th day of the tendon healing.

The larval salivary gland secretions ofBombus atratus were studied with cytochemical and cytophysical methods. In the young feeding larvae a proteic filamentous secretion depicting striations perpendicular to the long axis of its fibrillary threads and exhibiting a wellordered macromolecular array was found. It appears not to differ from the silk secretion described for the fully grown larvae of an europeanBombus species. However, in the fully grown larvae ofB. atratus changes which have not yet been reported for other bees occur involving the salivary gland secretion. Two secretion types are then distinguishable. One is composed of carboxylated and sulfated acid glycosaminoglycans and glycoprotein(s) (mucous secretion), and the other has the same composition as that of the filamentous secretion of the young larvae, although differing morphologically and in terms of macromolecular alignment (flocculent secretion). The filamentous secretion is assumed to be involved inB. atratus with the spinning of the “silken partitions” which at a relatively early stage separate the larvae reared within a common cell from one another. The mucous and flocculent secretions will participate in the cocoons which will cover the pupating larvae. The filamentous and flocculent secretions appear to contain an α-helical fibroin, glycoprotein(s) and lipoprotein(s), but not collagen-type proteins.

Summary Intrinsic (IB) and form or textural birefringence curves (FBC) were obtained measuring optical path differences in non stained sections of articular cartilages.Differences were not detected when comparing IB and FBC of the articular surface with those of the intermediate zone (Collagen II), but were found when these curves were compared with those of tendons (Collagen I).A detailed description of arrangements of collagen fibers are reported with no confirmation of Gothic arches.Hyaluronidase and papain digestion caused a decrease in birefringence of the cartilage supporting the conclusion that acid proteoglycans (APG) present molecular orientation in those structures.Histochemical data and histophotometric measurements confirmed that in articular cartilage there are variations in APG concentration and complexation with Collagen II from the articular surface (AS) down to the bone zone, AS displaying much more available — NH3+ groups.Molecular order in the articular cartilage optimizes the state of aggregation in its structures, and is the basis for its piezoelectric and mechanical properties.

Wistar rats were submitted to the action of active lectins from common dry beans (Phaseolus vulgaris) and from jack beans (Canavalia ensiformis, DC). Raw common bean was offered to the rats in an otherwise balanced diet to make 10% protein as the sole protein source. A single dose of 20 mg of jack bean lectin (concanavalin A) was given by gastric intubation. Half of the rats receiving raw bean died within 22 days of experiment. Histological findings showed ulceration and necrosis of the intestinal villi in the surviving rats. In some cases the lesions reached also the submucosa. Gastric intubation of concanavalin A caused intense scaling off in the apical portion of the villi.

Summary Form birefringence curves were determined for fixed (and unfixed rat axons before and after lipid extraction. The total detected birefringence was assumed to be due to the macromolecular array of myelin sheath components (phospholipids, cholesterol, and proteins). Unfixed nerves displayed negative form birefringence. Their form birefringence curve exhibited a = refractive index match point positioned at n = 1.46 (intrinsic birefringence). Formalin fixation induced decrease in the optical retardation values but did not affect the profile of the form birefringence curves. After lipid removal, however, the anisotropic patterns of the fixed and unfixed nerves changed. A positive form birefringence was then exhibited, which is attributed to the macro-molecular orientation of the protein framework of the myelin sheath. Changes in the shape of the form birefringence curve and in the localization (and number) of the refractive index match point were found. They varied as lipids had been removed from nerves subjected or not to fixation. Therefore, the form birefringence of the myelin sheath proteins plays a part in the total phenomen observed in the whole nerves interfering with that displayed by phospholipids and cholesterol of the mentioned structure.

An analysis of profiles of Feulgen hydrolysis curves has been carried out in two heterochromatic (B-15 and C-11) and one euchromatic (C-1) regions of polytene chromosomes of salivary glands of 66 day old Rhynchosciara americana larvae. Differences among these three regions have been found mostly concerning the period of predominant apurinic acid breakdown including the “lag” phase of the apurinic acid depolymerization. The hydrolysis time for which maximal Feulgen-DNA values are attained also differs when comparing the hetero- and euchromatic regions of the C chromosome and the heterochromatic regions C11 and B15 to each other. Different labilities of DNA to acid hydrolysis have therefore been demonstrated for these chromosome regions. This has been tentatively correlated with differences in non-histone proteins some of which are assumed to be involved in chromatin compactness. Others (probably not concerned with chromatin compactness) are suggested to be associated with certain specific DNA types which are known to occur in these chromosome regions.

Based on the results obtained on the grounds of the present discussion we may conclude: Under the described condition, in the detached tendons of guinea pigs and also in the implanted tendons the acid mucopolysaccharides gradually undergo a structural disorder, loosing its molecular orientation. In the referred tendons, the loss of structural orientation of the acid mucopolysaccharides proceeds the desorientation of the collagen polypeptide chains. In the tendon repair process, under the described conditions, acid mucopolysaccharides with oriented macromolecules are elaborated during the fibrillogenesis phenomenon.

The binding capacity of concanavalin A (Con A) to condensed euchromatin and heterochromatin was investigated in chicken erythrocyte nuclei (CEN), mouse liver cells, Zea mays mays meristematic cells and Drosophila melanogaster polytene chromosomes after 4 N HCl hydrolysis to determine whether binding was preferentially occurring in bands and heterochromatin. Dry mass (DM) variation was investigated in CEN by interference microscopy. Feulgen and Con A reactions were employed for all materials to correlate the loci of the two reactions. Quantifications and topological verifications were carried out by video image analysis (high performance cytometry). It was observed that 4 N HCl hydrolysis caused an important DM loss in CEN leaving a level corresponding to the average DNA DM content. In this material, Con A binding was restricted to the nuclear envelope, which reinforces the idea of the absence of a nuclear matrix in these cells. The other cell types exhibited a correspondence of Feulgen-positive and Con A-reactive areas. The Con A reaction was highly positive in the condensed chromatin areas and heterochromatin. This fact led us to speculate that Con A-positive proteins may play a role in the chromatin condensation mechanism, endowing this structure with physico-chemical stability towards acid hydrolysis and contributing to its rheological properties. A capacidade de ligação da concanavalina A (Con A) a regiões condensadas de eucromatina e heterocromatina foi investigada em núcleos de eritrócito de frango (CEN), hepatócitos de rato, células meristemáticas de Zea mays mays e em cromossomos politênicos de Drosophila melanogaster após hidrólise com HCl 4 N para determinar a ocorrência de ligação preferencial em bandas e heterocromatina. A variação da massa seca foi investigada em CEN por microscopia de interferência, e reações de Feulgen e Con A foram empregadas para todos os materiais para correlacionar os loci de ambas reações. As quantificações e verificações topológicas foram levadas a efeito por análise de imagens (citometria de alta performance). Foi observado que a hidrólise por HCl 4 N causou uma importante perda de massa seca em CEN, permanecendo um nível correspondente ao conteúdo médio de massa seca de DNA. Neste material a ligação com Con A foi restrita ao envelope nuclear, reforçando a idéia da ausência da matriz nuclear nessas células. Os demais tipos celulares exibiram áreas reativas de cromatina condensada e heterocromatina. Este fato permite especular o papel desempenhado por proteínas Con A-positivas no mecanismo de condensação cromatínica. Esta estrutura glicoprotéica contribuiria para uma maior estabilidade físico-química da cromatina condensada, especificamente da heterocromatina, e também para suas propriedades reológicas.

O colágeno é a forma funcional de um oligômero biológico, cujos monômeros são aminoácidos quirais. Tecidos ricos em colágeno são estruturas opticamente anisotrópicas, birrefringentes e não-lineares. O objetivo deste trabalho foi caracterizar o colágeno tipo I não irradiado e após irradiação com laser de He-Ne polarizado, utilizando a microscopia de polarização para investigação da birrefringência, e a geração de segundo harmônico (GSH) para análise da susceptibilidade não-linear. Doze ratos Wistar foram divididos em quatro grupos, de acordo com as idades: 34, 48, 62 e 76 dias. Os grupos receberam, respectivamente, 1; 5; 9 e 13 irradiações de D= 1J/cm2, durante 46s, no tendão de Aquiles que, aleatoriamente, foi escolhido ser o direito ou esquerdo. Os animais foram então sacrificados e os tendões extraídos para a preparação das amostras. Para a microscopia de polarização foram obtidos cortes com 8μm de espessura, e as birrefringências de forma e intrínseca foram avaliadas. Para a GSH foi utilizado um laser de Ti:Safira (150fs) com 76MHz de taxa de repetição, diâmetro do feixe φ= 40μm, comprimento de onda central λ= 800nm e Δλ= 10nm e Pp= 110mW. Um polarizador linear foi posicionado no caminho do feixe. Duas objetivas foram utilizadas para focalizar e coletar a radiação incidente e emergente da amostra, respectivamente. Um analisador, também linear, foi introduzido logo após a objetiva coletora. Um filtro absorvedor na faixa infravermelha do espectro eletromagnético, mas transmissor na faixa azul, foi utilizado para garantir que apenas λ= 400nm era medido. A razão, ρ, entre dois tensores de hiperpolarizabilidade independentes foi estudada. As amostras irradiadas apresentaram maior birrefringência (Δn) e maior susceptibilidade não-linear (ρ), em comparação com as não irradiadas. Estes resultados indicam que o laser de He-Ne polarizado alinha as fibrilas de colágeno ao longo eixo do tendão. The collagen is the functional form of a biological oligomer whose monomers are chiral amino acids. Tissues filled up of collagen are anisotropic optically structures, birefringent and nonlinear. The purpose of this study was characterizing nonirradiated and polarized He-Ne laser irradiated type I collagen using polarized light microscopy to investigate the birefringence and the second harmonic generation (SHG) to analyze the nonlinear susceptibility. Twelve Wistar rats were alienated into four groups according to the ages: 34, 48, 62 and 76 days. The groups received, respectively, 1; 5; 9; and 13 irradiations of D= 1J/cm2 during 46s on Achilles tendon, which was chosen randomly as right or left. Thereafter, the animals were sacrificed and the tendons were extracted to prepare the specimens. For polarized light microscopy, eight-μm sections were obtained to evaluate the form and intrinsic birefringences. For SHG, it was used a Ti:Safira (150fs) laser at a repetition rate of 76MHz, beam diameter of φ= 40μm, wavelength of λ= 800nm and Δλ= 10nm and Pp= 110mW. The beam passed through a linear polarizer and two microscope objectives were used to focalize and collect the incident and emergent radiation, respectively. A second linear polarizer was placed after of collecting objective. An infrared absorber and blue transmitter filter was used a sure that only λ= 400nm was measured. The ratio, ρ, between two hyperpolarizability tensors was studied. The irradiated samples presented higher birefringence (Δn) and nonlinear susceptibility (ρ) when compared to nonirradiated samples. These results indicate that a polarized He-Ne laser aligns the collagen fibrils to the long tendon axis.