The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined medium (modified Tyrode's solution) without glucose. When different concentrations of NaCl were added to the medium, the proportions of embryos developed to the >/=8-cell, morula and blastocyst stages 96, 144 and 192 h post insemination, respectively, were significantly higher at 89 to 114 mM than 64 to 76 and 126 to 139 mM NaCl. A high proportion (28%) of blastocyst-stage embryos 192 h post insemination was obtained at 89 mM NaCl. When calculated osmolarity in the medium with 64 mM NaCl was varied by adding D-sorbitol, significantly higher proportions of morula-stage embryos were obtained at 265 to 315 mOsm (27 to 38%) than 215 (9%) and 365 (2%) mOsm, but the development to the blastocyst stage was difficult at any osmolarities (215 to 365 mOsm) tested. In the medium with a fixed osmolarity (315 mOsm) but with different concentrations (64 to 114 mM) of NaCl, there were no differences in the proportions (29 to 33%) of morula-stage embryos among different NaCl concentrations. However, significantly higher proportions of embryos developed to the blastocyst stage at 89 to 101 mM (22 to 23%) than 64 to 76 (0 to 9%) and 114 (11%) mM NaCl. When Cl- concentration in the medium with 64 mM NaCl was adjusted by adding choline chloride, significantly higher proportions of embryos developed to the morula stage at 97 to 122 mM (32 to 40%) than 72 (6%) and 147 (2%) mM Cl-, but few embryos developed to the blastocyst stage at any Cl- concentrations (72 to 147 mM) tested. In the medium with 64 or 114 mM NaCl and each with 2 different Na (+)K (+) ratios, there were no differences in the proportions of morula- and blastocyst-stage embryos between different Na+ K+ ratios (31 and 39 at 64 mM NaCl, and 39 and 47 at 114 mM NaCl) at each NaCl concentration. When glucose was added to the medium with 89 mM NaCl 120 h postinsemination, there were no significant differences in the proportions (40 to 48%) of morula-stage embryos 144 h post insemination among different concentrations (0 to 6.95 mM) of glucose. The proportion (33%) of blastocysts 192 h post insemination at 2.78 mM glucose was significantly higher than the values at 0 (22%), 5.56 (19%) and 6.95 (15%) mM but not different compared with the values at 1.39 (23%) and 4.17 (28%) mM. In conclusion, NaCl concentration in a defined medium is one of the most important factors for the development of bovine embryo to the blastocyst stage, but the development of embryos up to the morula stage is also regulated by osmolarity and/or Cl-concentration.     

Chauhanellus Bychowsky & Nagibina, 1969 (Monogenea) from ariid fishes (Siluriformes) of Peninsular Malaysia

Twelve new species of Chauhanellus Bychowsky & Nagibina, 1969 have been found on six species of ariid from Peninsular Malaysia: Chauhanellus trifidus n. sp., C. digitalis n. sp., C. malayanus n. sp., C. forcipis n. sp. and C. intermedius n. sp. from Arius sagor; C. aspinous n. sp. from Arius venosus; C. caelatus n. sp. from Arius caelatus; C. auriculatum n. sp., C. poculus n. sp. and C. pulutanus n. sp. from Arius maculatus; C. duriensis n. sp. from Arius thalassinus; and C. osteogeneiosi n. sp. from Osteogeneiosus militaris. Some of these Chauhanellus species possess characteristics that are not commonly observed in the genus. C. aspinous n. sp., C. intermedius n. sp. and C. digitalis n. sp. exhibit features found in both Chauhanellus and Hamatopeduncularia: these include absence of spines on the mainpart of the dorsal anchors in C. aspinous n. sp. and C. intermedius n. sp. and presence of haptoral digitation in C. digitalis n. sp. Other features are the five transverse rows of peduncular spines in C. duriensis n. sp., ear-like projections on the anchors in C. auriculatum n. sp., and thin sclerotised plates that partly envelope the ventral anchors in C. forcipis n. sp. Mid-dorsal appendices occur on the dorsal bars of seven of the present species.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Cell signaling and heat shock protein expression

Exposure of cells and organs to heat shock is associated with numerous changes in various cellular metabolic parameters and overexpression of proteins collectively known as heat shock proteins (HSP). In this communication we review the cell-signaling events that are altered in response to heat shock as they relate to the subsequent induction of HSP 70 kd (HSP-70) expression. We also review the mechanisms by which HSP-70 is involved in conferring cytoprotective effects. The possibility of altering HSP expression through manipulations of the cell-signal process has clinical importance.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or Department of Defense.     

Neocalceostoma Tripathi, 1957 and Neocalceostomoides Kritsky,Mizelle & Bilqees, 1978 (Monogenea: Neocalceostomatidae n. fam.) from ariid fishes of Peninsular Malaysia

Three new species of Neocalceostomoides Kritsky, Mizelle & Bilqees, 1978 and Neocalceostoma elongatum Tripathi, 1957 were found on five species of Arius from Peninsular Malaysia. N. elongatum was collected from Arius sagor. A. venosus and A. maculatus, whereas Neocalceostomoides spinivaginalis n. sp., N. hamatum n. sp. and N. simplex n. sp. were obtained from A. thalassinus, A. sagor and A. caelatus, respectively. The three new species of Neocalceostomoides are similar to Neocalceostoma elongatum in the morphology of the soft anatomical parts and in having 14 marginal hooks, but differ in having the two anchors situated far apart without a connecting bar. The possibility that Neocalceostomoides and Neocalceostoma are synonymous is noted. The generic diagnosis of Neocalceostomoides is amended herein and a new family, the Neocalceostomatidae, is proposed to accommodate Neocalceostoma and Neocalceostomoides.