Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.     

Responses against complex antigens in various models of CD4 T-cell deficiency

The most common models of CD4 T-cell deficiency are mice exogenously injected with anti-CD4 antibody (Ab), CD4 knockout (CD4^−/−) and major histocompatibility complex (MHC) class II knockout (class II^−/−) mice. We recently described the anti-CD4 Ab transgenic mouse (GK) as an improved CD4 cell-deficient model. This review compares this new GK mouse model with the widely available class II^−/− and CD4^−/− mice, when exposed to complex antigens (foreign grafts and during bacterial or viral infection). We highlight here the cytometric and functional differences (including Ab isotype, viral or bacterial clearance, and graft survival) among these CD4 cell-deficient models. For example, whereas grafts are generally rejected in class II^−/− and CD4^−/− mice as quickly as in wild-type mice, they survive longer in GK mice. Also, CD4^−/− mice produce IgG against both simple model and complex antigens, but class II^−/− and GK mice produce small amounts of IgG2a against complex antigens but not simple model antigens. These differences harbinger the caveats in the use of these various mice.     

Colchester revisited: a genetic study of mental defect.

This reanalysis of a classic survey leads to inferences about design of genetic studies, resolution of heterogeneity, and the role of autosomal and sex-linked genes in mental retardation, which is no longer refractory to segregation analysis. By discriminating between sociofamilial and biological types we estimate that at least 351 autosomal loci can produce mental retardation, with an inbred load of 0.83 detrimental equivalents and a mutation rate of 0.008 per gamete, or less than 2.4 X 10(-5) per locus. The distribution of probands was estimated as: 7 per cent medical, 60 per cent sociofamilial, and 33 per cent biological. Simple genetic mechanisms account for virtually all the biological category. Within the sociofamilial group cultural inheritance and polygenes could not be resolved.     

Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.      

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

Identification of dermatophyte species by 28S ribosomal DNA sequencing with a commercial kit

We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex.     

Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2.

We have cloned four cyclin-B homologs from Saccharomyces cerevisiae, CLB1-CLB4, using the polymerase chain reaction and low stringency hybridization approaches. These genes form two classes based on sequence relatedness: CLB1 and CLB2 show highest homology to the Schizosaccharomyces pombe cyclin-B homolog cdc13 involved in the initiation of mitosis, whereas CLB3 and CLB4 are more highly related to the S. pombe cyclin-B homolog cig1, which appears to play a role in G1 or S phase. CLB1 and CLB2 mRNA levels peak late in the cell cycle, whereas CLB3 and CLB4 are expressed earlier in the cell cycle but peak later than the G1-specific cyclin, CLN1. Analysis of null mutations suggested that the CLB genes exhibit some degree of redundancy, but clb1,2 and clb2,3 cells were inviable. Using clb1,2,3,4 cells rescued by conditional overproduction of CLB1, we showed that the CLB genes perform an essential role at the G2/M-phase transition, and also a role in S phase. CLB genes also appear to share a role in the assembly and maintenance of the mitotic spindle. Taken together, these analyses suggest that CLB1 and CLB2 are crucial for mitotic induction, whereas CLB3 and CLB4 might participate additionally in DNA replication and spindle assembly.     

Glycoproteins present in human follicular fluid that inhibit the zona- binding capacity of spermatozoa

Previous studies have suggested that human follicular fluid contains factors that reduce the zona-binding capacity of spermatozoa. The present study provides further evidence of the existence of such factors. Using the hemizona binding assay (HZA), we have shown that the inhibitory effect of human follicular fluid on the zona-binding capacity of spermatozoa is concentration-dependent, an inhibitory effect being detected when the concentration of human follicular fluid was > or = 10%. A 1% concentration of human follicular fluid did not possess this inhibitory activity. Heating human follicular fluid at 56 degrees C for 30 min did not affect its inhibitory properties; treatment with proteinase-K abolished such inhibition. Human follicular fluid was fractionated sequentially by concanavalin-A affinity chromatography, Mono Q ion-exchange chromatography and Superose-12 gel filtration. The zona binding inhibitory activity resided in the fraction which bound to the lectin and Mono Q column and contained molecules with native molecular weights of 32 and 192 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis suggested that the 192 kDa glycoprotein was a tetramer, while the 32 kDa glycoprotein remained as a single molecular species under denaturing conditions. We conclude that two glycoproteins were responsible for the zona binding inhibitory activity of human follicular fluid. The physiological role of these factors remains unclear.