Análise citológica e cariométrica da ação da colchina sôbre a espermatogênese dos hemípteros

The action of colchicine upon the spermatogenesis of Triatoma infestans, (Hemipt. Heteroptera), has been studied and the different categories of giant spermatids that appear during the treatment have been compared with the nuclear volumes of the whole series of normal spermatogenetic stages. The following facts have been ascertained: 1) 4 hours after the treatment the gonial mitotic metaphases, and the 1st. and 2nd. metaphases of meiosis are stopped. The prophasic stages of meiosis and diakynesis appear to be normal. After 9 days of treatment, all the tetrads are broken in the meiotic metaphases and the cells appear with 44 and 22 chromosomes respectively, scattered in the cytoplasm. 2) At 9 days, practically all spermatogenetic stages have disappeared except for a few cysts of spermatogonia, and practically the whole testicle is full of cysts of spermatozoa and spermatid, with some large zones of necrosis with pycnotic nuclei. The spermatids appear to be of different sizes and the statistical analysis of the nuclear volumes gives a polymodal hystogram with 4 modes, whose volumes are in the ratio of 1:2:4:8. Ripe spermatozoa seem to have a certain volume variability, that has not been possible to analyse quantitatively. All these facts confirm what DOOLEY found in the colchicinized Orthoptera testicle. 3) The caryometric analysis conducted statistically on the normal stages of the spermatogenesis (resting spermatogonia, gonial prophases, leptotene, "confused stage", diakynesis, and spermatid) revealed the following facts: a) Considering the volume of the resting, spermatogonia as 1, their mitotic prophases have a volume of 2. Some rare prophases appear to have a volume of 4 and probably belong to tetraployd spermatogonia normally present in the testicle of Hemiptera. b) The first spermatocyte at the beginning of the auxocitary growth (leptotene) has a volume of 2, which is equal to that of them gonial prophase. It grows further during the "confused stage" and reduplicates, reaching thus the volume of 4. Diakynesis has a rather variable nuclear volume and it is higher than volume 4. This is probably of physico-chemical nature and not a growth increase. c) The spermatid at the beginning of the spermiogenetic process has a volume of 1 which is very constant and homogeneous. 4) These results can be summarized concluding that the meiotic process begins from a spermatogonium at the end of his mitotic interphasic growth (vol. 2) and instead of entering into the mitotic prophase transforms itself into the leptotene spermatocyte. During the diplotene ("confused stage") the volume of the nucleus doubles once more and reaches volume 4. In consequence of the two successive meiotic divisions the spermatid, although having an haploid number of chromosomes, has a nuclear volume of 1, just like the diploid spermatogonium. The interpretation of this strange result probably comes from the existence of the "tertiary split" in the chromosomes of the haploid set, that has been illustrated in the Hemiptera by HUGUES SCHRADER and in Orthoptera by MICKEY and co-workers. The tertiary split indicates that the chromosomes of the haploid set are constituted from almost two chromonemata, and this double constitution corresponds to the double cycle of reduplication that takes place during the spermatogenesis starting from the resting gonia. In Triatoma infestans the tertiary split appears in the chromosomes in the 1st. and 2nd. metaphases and in the diakynesis. In the blocked metaphases at the 9th. day of colchicinization some of the 44 elements scattered in the cytoplasm, show, when properly oriented, the split very clearly. Some new and strange facts revealed by SCHRADER and LEUCHTEMBERGER in Arvelius suggest the possibility of other interpretations of the rhythmic growth in special cases. There appears the necessity of more knowledge about the multiple or simple constitution of the chromosomes in somatic and spermatogonial mitosis.

Morfologia do Mycobacterium leprae hominis e do M. leprae muris: estudo baseado na microscopia electrônica e de contraste de fases

Hansen's Bacillus: By electron microscopy this bacillus shows membrane and halo, this being more visible when sorrounding the globi or bundles of bacilli; shows, also, free granules of various sizes which were before considered as dust of the dyes; shows external granules bound with the membrane and some times branching. By phases contrast microscopy examining leproma suspensions and subcataneous lymph at 400 x we saw many free granules with intense rotatory movement; granulated bacilli with screw, skip or stroke motion, producing slow progressive motion. All such elementes are surrounded by a halo, corresponding to the classical gloea. By a patient and delayed examination we were able to see that the internal granules are motile and help the progression of the bacilli, giving the impression that the cytoplasm is liquid. By a lasting observation we could see the larger granules form prolapse, like a pseudopode and abandon the bacilli and going in very rapid rotatory movement. There are branched bacilli; there are pedunculated fred granules like comets. The addition of a drop of formol at the preparation stops all movements. Stefansky's Bacillus: Repeated examination by RCA electron microscope, type EMU-25 of fresh suspensions of rat lepromas, led us to confirm the close relationship between human and murine leprosy agents. We examined also material from carabo (Lepra bubalorum) from Java, but due to fixation, the material was unsuitable for comparative studies. The Stefansky's bacilli showed also emmbranes and halos, internal or external granules (smaller than those of Hansen's bacillus). The bacilli shaded by chromium look thicker and shorter than those of Hansen. Due to electron bombardment both, Hansen's and Stefansky's baccilli suffer considerable alterations in their structure, showing black barrs of chromatin condensation at their extremities as also in their centers. By phase microscopy the Stefansky's bacilli showed elements with 1, 2 (bipolar), 3 or more internal small granules, developing identical movements as those of Hansen. The globi seem to be non-motile but the free bacilli appearing around the globi show intense movement. At 1000 x the examination is less satisfactory than at 400 x. The addition of formol solution in the preparation suppresses all movements, even the brownian, but the material becomes more suitable for the study of static morphology of the bacilli. CONCLUSION - The electron and phases contrast microscopy of leprous material from different types and phases of the disease may explain some of the unknown aspects of the biology and morphology of the bacilli.

A study of the endocervical columnar cells I - morphology and frequency

After the observation of many thousands of histological sections of the endocervical mucosa it became evident that its columnar cells present a great variety of aspects not only those of the surface of the canal but also those of the glands. A classification of these cells was made taking into account the staining affinity, the intensity staining of the cytoplasm, the presence or absence of cilia, the shape and location of the nucleus. The various combinations of these different data made possible the characterization of 26 types of cells which we labelled by the alphabetical letters. Two hundred and fifty cervices obtained by cervical amputation and by hysterectomy were studied. The uteri presented lesions in the course of routine laboratory examination. In each of the 250 histological sections there were specifically counted 2,000 columnar cells which cover the cervical canal and 2,000columnar cells which form the glands. A graphic representation of the frequency of both the superficial and glandular columnar cells was presented; this was given the name EPITHELIOGRAM. The variation of the cellular "composition" of each epithelium is discussed and the frequency of the various cellular types after the count of one million of cells is presented.

The viral nature of Toddia França, 1912

Toddia França, 1912 under the light microscope occurs as inclusion corpuscles in the cytoplasm of erythrocytes of cold-blooded vertebrates sometimes accompanied by crystalloid bodies. Its position among the protozoans or the viruses has been discussed by some authors, but remained unclear. To elucidate this problem we studied Toddia from a Brazilian frog (Leptodactylus ocellatus) by electron microscopy. In the cytoplasm of the infected cells we found no protozoan, but rather virus-like particles often hexagonal in outline, averaging 195 nm excluding their two involving membranes, and presenting a central area of variable electron density. Particles at different stages of development were generally found around or on area lighter density than the cytoplasm. which resembled a virus synthesis site. At high magnification, the nuclear or cytoplasmic crystals allied to Toddia resembled the crystalline lattice of the inclusion bodies associated with the polyhedrosis viruses and poxviruses from insects, of the capsules of granulosis viruses and of other protein crystals in ultrathin sections. Cytochemical tests in Toddia corpuscles displayed exclusively the presence of deoxyribonucleic acid. These findings indicate that Toddia is not a protozoan and demonstrate that it is in all probability a viral inclusion corpuscle. Taking into account the nucleic acid type found in its structure (DNA) and the hexagonal shape usually shown in ultrathin sections by its component particles, which have a cytoplasmic site of synthesis and assembly, we tentatively relate Toddia with the so-called "Icosahedral Cytoplasmic Deoxyriboviruses". We believe that the present paper gives the first report of virus-like particles in L. ocellatus.

Malignant transformation of a rat fibroma by the treatment with an anti-fibrosing drug: CY-168F (Plastenan)

Fifteen albino (Sprague Dawley) rats with subcutaneous transplanted fibromas was used in the present study. The tumour was formed by typical fibroblasts in a dense collagen matrix and was provenient from a fibroma that appeared spontaneously in an albino rat of the same strain. Ultrastructurally collagen disclosed normal periodicity and the fibroblasts showed irregular notched nuclei with irregular distribution of chromatin, that suggests transitional aspects to fibrosarcoma. The 15 animals, from different passage groups, were divided into: 8 animals submitted to treatment with the drug acexamic acid (CY-168F) - N acetyl-amino-6-hexanoic acid (plastenan) and 7 untreated control animals. Three of the treated animals showed a malignant transformation to fibrosarcoma. transitional histological features from typical fibroma to highly indifferentiated fibrosarcoma could be detected in come animal subjected to repeated biopsies. Ultrastructural study disclosed nuclear alterations and hyperactive ergastoplasm and collagen containing inclusions into the cytoplasm of fibroblasts. In the group of 7 untreated naimals, no malignant transformation could be detected histologically. Two aspects deserve attention: the malignant potential of a typical fibroma and the apparent effect of an antifibrosing drug in inducing malignization of this tumour.

The hemocytes of Panstrogyllus Megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.

The potassium impermeable apical membrane of insect epithelia: a target for development of safe pesticides

Columnar cell apical membranes (CCAM) in series with goblet cell apical membranes (GCAM) form an electroosmotic barrier separating the midgut lumen from epithelial cell cytoplasm. A unique K+ ATPase in GCAM generates three gradients across this barrier. A greater than 180 mV electrical gradient (lumen positive) drives amino acid uptake through voltage-dependent K+ symports. A greater than 1000-fold [H+] gradient (lumen alkaline) and a greater than 10-fold [K+] gradient (lumen concentrated) are adaptations to the high tannin and high K+ content, respectively, in dietary plant material. Agents which act on the apical membrane and disrupt the PD, H+, or K+ gradients are potential insecticides. Insect sensory epithelia and mammalian stria vascularis maintain similar PD and K+ gradients but would not be exposed to ingested anti-apical membrane insecticides. Following the demonstration by Sacchi et al. that Bacillus thuringiensis delta-endotoxin (Bt) induces specifically a K+ conductance increase in CCAM vesicles, we find that the K+ channel blocking agent, Ba2+, completely reverses Bt inhibition of the K+-carried short circuit current in the isolated midgut of Manduca sexta. Progress in characterizing the apical membrane includes finding that fluorosulfonylbenzoyladenosine binds specifically to certain GCAM polypeptides and that CCAM vesicles can be mass produced by Ca2+ or Mg2+ precipitation from Manduca sexta midgut.

Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduvidae)

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrogylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, espcieally rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER).They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrondensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrondense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasme is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/cubic millimeter of hemolymph in the 4th intar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.

Action of colchicine on hepatic schistosomal granuloma

Amorphous material and altered collagen fragments within dilated secretory vesicles and cisternae of fibroblast cytoplasm were the main ultrastructural changes seen in hepatic periovular granulomas formed in mice infected with Schistosoma mansoni and treated with colchicine. Despite promoting ultrastructural changes in the fibroblasts found in hepatic periovular granulomas, colchicine administration to infected mice did not significantly change the light microscopic appearance of the hepatic schistosomal lesions, did not diminish the amount of total hepatic collagen, and did not change the collagen isotypes in the granulomas, as observed after a comparative study with non-colchicine treated infected control mice. When administered to mice two weeks after curative treatment of schistosomiasis with praziquantel, colchicine did not seem to increase extracellular collagen degradation or to induce a more rapid resorption of hepatic periovular granulomas, although still promoting ultrastructura alterations in fibroblasts.

Ultrastructure of the ovary of Dermatobia hominis (Diptera: cuterebridae). I. Development during the 3rd larval instar

The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrodense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.

Ultrastructural and stereological analysis of trypanosomatidis of the genus Endotrypanum

Culture forms of four strains of Endotrypanum (E. schaudinni and E. monterogeii) were processed for transmission electron microscopy and analyzed at the ultrastructural level. Quantitative data about some cytoplasmic organelles were obeined by stereology. All culture forms were promastigotes. In their cytoplasm four different organelles could be found: lipid inclusions (0,2-0,4 µm in diameter), mebrane-bounded vacuoles (0.10-0,28 µm in diameter), glycosomes (0,2-0,3 µm in diameter), and the mitochondrion. The kenetoplast appears as a thin band, except for the strain IM201, which possesses a broader structure, and possibly is not a member of this genus. Clusters of virus-like particles were seen in the cytoplasm of the strain LV88. The data obtained show that all strains have the typical morphological feature of the trypanosomatids. Only strain IM201 could be differentiated from the others, due to its larger kenetoplast-DNA network and its large mitochondrial and glycosomal relative volume. The morphometrical data did not allow the differentiation between E. schaudinni (strains IM217 and M6226) and E. monterogeii (strain LV88).

Ultrastructure of Giardia duodenalis trophozoites group from hamster: some relevant aspects

Throphozoites of Giardia duodenalis group obtained from fragments or scratched of hamster's mucosa were examined by transission electron microscopy. The fine structure of the trophozoites are presented and comapred with those described for other animals. Some of the trophozoites present the cytoplasm full of glycogen, rough endoplasmic reticulum-like structures and homogeneous inclusions not enclosed by membranes, recognized as lipid drops, which had not been observed in Giardia from other animals. The adhesive disk is composed of a layer of microtubules, from which fibrous ribbons extend into the cytoplasm; these ribbons are linked by layer of crossbridge filaments that shows an intermediary dense band, described for the first time in this paper. The authors regard this band as the result of the cross-bridge filaments slinding in the medium region between adjacent fibrous ribbons, and suggest a contractile activity for them. The role of the adhesive disk on the trophozoite mechanism of attachment to host mucosa is also discussed.

Recent advances in pharmacologic study of natural anticancer agents in China

In this paper a number of anticancer agents of natural origin will be presented. Hydroxycamtothecin (HCPT) was found to produce a strong inhibitory action on a variety of animal tumors. It is also effective for treatment of patients with gastric carcinoma, liver carcinoma, tumor of head and neck or leukemia. Pharmacologic studies showed that it could depress S phase of tumor cells significantly and cause formation of cellular chromatid breaks. By means of alkaline elution and nick translation methods it has been proved that HCPT induced DNA singlo strand breaks remarkably. Homoharringyonine (hhrt) was shown to be effective against acute leukemia. Recent experiments in tumor-bearing mice inidcated that (HHRT) could diminish tumor metastasis. Using molecular hybridization technique it was demonstrated that (HHRT) decreased the content of c-myc RNA in the cytoplasm but not in the nuclei. Lycobetaine (LBT) poddrddrf dytnh inhibitory effects on a number of ascites tumors. In clinical trials it was against ovarian and gastric carcinomas. It is able to intercalate into DNA. Oxalysine (OXL) is a new antibiotic and shown to be effective against tumor metastatis. When used in combination with 5-FU, its anticancer action could be enhanced. Other natural compounds such as indirubin, ß-elemene, irisquinone, oridonine, norcantharidin and PSP have been also found to possess antitumor action.

Plant lectins, chemical and biological aspects

Lectins, carbohydrate-binding proteins of non-immune origin, that agglutinate cells or precipitate polysaccharides and glycoconjugates, are well distributed in nature, mainly in the Plant Kingdom. The great majority of the plante lectins are present in seed cotyledons where they are found in the cytoplasm or int he protein bodies, although they have also been found in roots, stems and leaves. Due to their peculiar properties, the lectins are used as a tool both for analytical and preparative purposes in biochemistry, cellular biology, immunology and related areas. In agriculture and medicine the use of lectins greatly improved in the last few years. The lextins, with few exceptions, are glycoproteins, need divalent cations to display full activity and are, in general, oligomers with variable molecular weight. Although the studies on lectins have completed a century, their role in nature is yet ynknown . Several hypotheses on their physiological functions have been suggested. Thus, lectins could play important roles in defense against pathogens, plant-microorganism symbiosis, cell organization, embryo morphogenesis, phagocytosis, cell wall elongation, pollen recognition and as reserve proteins. A brief review on the general properties and roles of the lectins is given.

Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila

The blood cells of the pulmonate snail Biomphalaria tenagophila, an important transmiter of the trematode Schistosoma mansoni in Brazil, were examined by ligth and transmission electron microscopy (TEM). Two hemocyte types were identified: hyalinocytes and granulocytes. Hyalinocytes are small young (immature), poorly spreading cells, which have a high nucleocytoplasmic ratio and are especially rich in free ribosomes. They do not appear to contain lysosome-like bodies and represent less than 10% of the circulating hemocytes. Granulocytes are larger hemocytes which readily spread on glass surface and which strongly react to the Gomori substrate, indicating the enzyme acid phosphatase usually found in lysosomes. Ultra-structurally, they contain a well-developed rough endoplasmic reticulum, dictyosomes and some some lysosome-like dense bodies. Granulocytes do not exhibit a characteristic granular aspect and the few granules observed in the cytoplasm should correspond to a lysosome system. They were named granulocytes instead of amoebocytes to use the same terminology adopted for Biomphalaria glabrata in order to make easier comparative studies. This is a preface study for more specific investigations on the functional activities of the blood cells of B. tenagophila and their interactions with the trematode parasite.