Phylogeny of novel naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea revised

Naked filose and reticulose protozoa were long lumped as proteomyxids or left outside higher groups. We cultivated eight naked filose or reticulose strains, did light microscopy, 18S rDNA sequencing and phylogeny (showing all are Cercozoa), and sequenced 80 environmental 18S-types. Filose species belong in subphylum Filosa and reticulose ones in subphylum Endomyxa, making proteomyxids polyphyletic. We therefore transfer the classically mainly reticulose Proteomyxidea to Endomyxa, removing evident filosans as new class Granofilosea (including Desmothoracida, Acinetactis and new heliomonad family Heliomorphidae (new genus Heliomorpha (=Dimorpha)). Five new species of Limnofila gen. n. (L. mylnikovi; L. anglica; L. longa; L. oxoniensis; L. borokensis, previously misidentified as Biomyxa (=Gymnophrys) cometa) form a large freshwater clade (new order Limnofilida). Mesofila limnetica gen., sp. n. and Nanofila marina gen., sp. n. group separately in Granofilosea (Cryptofilida ord. n.). In Endomyxa, a new genus of reticulose proteomyxids (Filoreta marina, F. japonica, F. turcica spp. n., F. (=Corallomyxa) tenera comb. n.) forms a clade (Reticulosida) related to Gromiidea/Ascetosporea. Platyreta germanica gen., sp. n. and Arachnula impatiens are related vampyrellids (Aconchulinida) within a large clade beside Phytomyxea. Biomyxidae and Rhizoplasmidae fam. n. remain incertae sedis within Proteomyxidea. Gymnophrydium and Borkovia are revised. The reticulose Corallomyxa are unlike Filoreta and possibly Amoebozoa, not Cercozoa.     

Differentiation of human embryonic stem cells to hepatocyte-like cells on a new developed xeno-free extracellular matrix

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated “RoGel,” which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC–HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.     

Scanning electron-microscopic study of sperm retention and migration in the vagino-cervical region of the rabbit

Summary The pattern of sperm retention and migration in the vagino-cervical region of rabbit was studied by means of scanning electron microscopy. The following phenomena were observed: 1) Spermatozoa located in the vagina and at the orifice of the ectocervix are usually distributed diffusely. They appear to be resting on the epithelial surface; many are structurally abnormal or decapitated. 2) The great majority of spermatozoa, however, seems to be anchored or retained in narrow epithelial channels with their heads in close file formations. This phenomenon was observed particularly in the fornix vaginae as late as 24 h post coitum. 3) A great number of spermatozoa invading the cervix evidently migrates in groups along the mucosal surface. Their heads are oriented toward the uterus and contact the epithelial cells. Spermatozoa that migrate beyond the cervico-uterine junction are distributed in the same manner. 4) Spermatozoa colonizing the cervical crypts appear to be attached via the anterior margins of their heads to the epithelial cells or to the tips of kinocilia. Their tails project into the crypt lumen. It is suggested that mainly three factors may be responsible for these phenomena: (i) the fact that only motile spermatozoa overcome the vagino-cervical barrier; (ii) the tendency of spermatozoa to move along the mucosa in close vicinity to the epithelial cells; and (iii) the inability to recognize mechanical barriers on the migration route (e.g., cervical crypts) and to overcome them quickly. This may be one of many possible causes leading to sperm retention in the vagino-cervical region.Supported by a grant from the Deutsche Forschungsgemeinschaft     

The effect of the trypsin inhibitor aprotinin (Trasylol) and TLCK on the gelatinolytic activity of acrosin and the motility of rabbit sperm in vitro

In a series of experiments the influence of the trypsin inhibitors aprotinin (Trasylol) and TLCK (N-p-tosyl-L-lysin chloromethyl ketone) on the gelatinolytic activity of acrosin and motility of rabbit spermatozoa was tested. Ejaculated, highly motile spermatozoa were washed in Brackett-Medium. 12.5 to 1000 microns Aprotinin and 50 to 1000 micrograms TLCK, respectively, were added to samples of 1 ml sperm suspension: the specimens were incubated at 37 degrees C. Increasing aprotinin concentrations reduced the gelatinolytic activity of acrosin and the sperm incubation at a concentration of 1000 micrograms Aprotinin/ml sperm. Spermatozoa in all TLCK specimens were entirely immotile 1.5 hours after incubation. The gelatinolytic activity of acrosin was obviously not inhibited at any TLCK concentration. These results suggest that, under these experimental conditions, aprotinin and TLCK may impair primarily the motility spermatozoa.     

Leishmania species: fatty acid composition of promastigotes

The fatty acid composition of the total lipid fractions of five different Leishmania organisms grown on Eagle's medium was determined by gas chromatography. The major fatty acids identified in the total lipid fractions of L. donovani, L. tropica major, L. tropica minor, L. tropica (England strain), and L. enriettii were C_12:0, C_13:0, C_14:0, C_15:0, C_16:0, C_17:0, C_18:0, C_18:1, C_18:2, and C_18:3. The statistical differences among the fatty acid methyl esters of different Leishmania organisms are discussed.Gas chromatographic analysis of the fatty acid methyl esters of the total lipid fractions of the original Eagle's medium and the media after harvesting of various Leishmania species revealed the presence of C_18:3 fatty acid in the total lipid fraction of the medium of L. donovani and the complete absence of 18-carbon unsaturated fatty acids in the total lipid fraction of the medium of L. enriettii. The use of such differences in the differentiation of various Leishmania species is discussed.     

The effect of trypsin inhibitors aprotinin (Trasylol) and TLCK administered locally by osmotic pumps on the gelatinolytic activity of acrosin and the transport of sperm cells in the female reproductive tract of rabbits

The trypsin inhibitors aprotinin (Trasylol) and TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone) were administered continuously into the lumen of the cervix uteri of sexually mature rabbits by means of surgically implanted osmotic minipumps. The doses were inseminated six days after implantation of the pumps, then sacrificed two to six hours after insemination and their reproductive tracts were prepared for gelatin substrate film test and scanning electron microscopy. At a pumping rate of 50 to 100 micrograms aprotinin/h neither gelatinolytic activity of acrosin nor sperm transport were visibly inhibited. TLCK, at a pumping rate of 10 micrograms/h, did not influence the proteolytic activity of acrosin; however it seemed, presumably for its toxicity, to destroy the fine structure of epithelial surfaces in the vagino-cervical region and to impair sperm transport. These results suggest, that acrosin, under these experimental conditions, is not inhibited by aprotinin and TLCK in vivo and may play no immediate role in sperm transport in the female reproductive tract.     

Diagnosis of calcium pyrophosphate dihydrate deposition disease by fine needle aspiration biopsy: a case report

BACKGROUND: Calcium pyrophosphate dihydrate deposition disease is a relatively rare disease with variable clinical presentations. CASE: A 73-year-old man presented with worsening lower back pain and fever. Fine needle aspiration biopsy of the lumbar vertebral bodies (L3-L4) revealed abundant neutrophils admixed with small, birefringent, rhomboid crystals in Diff-Quik-stained smears. These crystals were confirmed as calcium pyrophosphate dihydrate on cell block sections. A diagnosis of osteomyelitis and calcium pyrophosphate dihydrate deposition disease was rendered. The patient was treated with antibiotics and responded well. CONCLUSION: Calcium pyrophosphate dihydrate deposition disease can be diagnosed by fine needle aspiration biopsy, and an accurate diagnosis can be greatly facilitated by cell block sections. However, such a diagnosis may be neglected if the specimen is not carefully inspected.