Rates of assembly and degradation of bacterial ice nuclei

The kinetics of ice-nucleus assembly from newly synthesized nucleation protein were observed following induction of nucleation gene expression in the heterologous host Escherichia coli. Assembly was significantly slower for the small proportion of ice nuclei active above -4.4 degrees C; this was consistent with the belief that these nuclei comprise the largest aggregates of nucleation protein. The kinetics of nucleus degradation were followed after inhibiting protein synthesis. Nucleation activity and protein showed a concerted decay, indicating that most of the functional ice nuclei are in equilibrium with a single cellular pool of nucleation protein. A minority of the ice nuclei decayed much more slowly than the majority; presumably their nucleation protein was distinct either by virtue of different structure or different subcellular compartmentalization, or because of its presence in a metabolically distinct subpopulation of cells.     

Conserved repeats in diverged ice nucleation structural genes from two species of Pseudomonas

Sequence analysis shows that an ice nucleation gene (inaW) from Pseudomonas fluorescens is related to the inaZ gene of Pseudomonas syringae. The two genes have diverged by many amino acid substitutions, and have effectively randomized the third bases of homologous codons. By reference to their potential for change, it is shown that certain conserved features must have been maintained by selection pressure. In particular, their conservation of internal sequence repetition, with three orders of repeat periodicity in each gene, suggests that the pattern of repetition is significant to the gene products' function. We propose models for the structure of the gene products in which each order of periodicity would be required for the nucleation function.     

Ultrastructural localization of herpes simplex virus RNA by in situ hybridization

We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

Polyclonal carcinoembryonic antigen staining in the cytologic differential diagnosis of primary and metastatic hepatic malignancies.

In order to assess the utility of immunocytochemical staining of bile canaliculi with a polyclonal antiserum to carcinoembryonic antigen (pCEA) in the differentiation of primary hepatocellular carcinomas from metastatic malignancies, pCEA staining was performed on fine needle aspiration specimens from hepatic lesions in 60 patients. The original cytologic diagnoses were hepatocellular carcinoma in 22 patients, metastatic neoplasm or cholangiocarcinoma in 27 patients and benign hepatocytes in 11 cases. The cytologic diagnoses of malignancy were confirmed by surgical excision, autopsy or clinical investigations in 82% of the patients. Follow-up data, supported by pCEA staining, reversed the original cytologic diagnosis in three cases. Bile canalicular pCEA staining was identified in 18 of 22 cases of hepatocellular carcinoma and in all 11 benign hepatocellular aspirates. All 27 cases of metastatic malignancy or cholangiocarcinoma were negative for canalicular pCEA staining, although 11 cases exhibited cytoplasmic staining. Interpretation of pCEA staining was not affected by the intermingling of malignant cells and benign hepatocytes. Predictive values were 100% for a positive test and 87% for a negative test. These findings indicate that staining with pCEA antiserum is a useful adjunct in the differential cytologic diagnosis of malignant hepatic lesions.     

Biodecolourization of azo and triphenylmethane dyes by <Emphasis Type="Italic">Dichomitus squalens</Emphasis> and <Emphasis Type="Italic">Phlebia</Emphasis> spp

Nine white-rot fungal strains were screened for biodecolourization of brilliant green, cresol red, crystal violet, congo red and orange II. Dichomitus squalens, Phlebia fascicularia and P. floridensis decolourized all of the dyes on solid agar medium and possessed better decolourization ability than Phanerochaete chrysosporium when tested in nitrogen-limited broth medium. Journal of Industrial Microbiology & Biotechnology (2002) 28, 201–203 DOI: 10.1038/sj/jim/7000222 Received 12 July 2001/ Accepted in revised form 22 October 2001     

Membranes in the miotic apparatus of barley cells

Membranes in the mitotic apparatus have been investigated ultrastructually in dividing cells of barley (Hordeum vulgare). After osmium tetroxide- potassium ferricyanide or ferrocyanide postfixation (OsFeCN) of material that had been fixed in glutaraldehyde in the presence of Ca(++), the nuclear envolope (NE)-endoplasmic reticulum (ER) complex is selectively stained, permitting observations on the cellular pattern and structural ramifications of this membrane system that have not been previously recognized. Specifically, it is observed that membrane system that have not been previously recognized. Specifically, it is observed that during mitosis the NE-ER forms a continuous membrane system that ensheathes and isolates the mitotic apparatus (MA). Elements of ER progressively accumulate in the region of the spindle pole, becoming most concentrated by early anaphase. Within the MA itself, there are striking spindle- membrane associations; in particular, tubular elements of predominantly smooth NE-ER invade the spindle interior selectively along kinetochore microtubules. The membrane elements at the pole and surrounding the MA consist of tubular reticulum and fenestrated lamellae. Membranes of the MA thus resemble in considerable detail the tubular network and fenestrated elements of the sarcoplasmic reticulum of muscle. It is suggested that the NE-ER of the dividing barley cell may function in one or both of the following ways: (a) to control the concentration of free Ca(++) in the MA and (b) to serve as an anchor to chromosome motion.     

Effects of Erythropoietin in Murine-Induced Pluripotent Cell-Derived Panneural Progenitor Cells

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1–3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of β-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.     

A Cross-Sectional Randomised Study of Fracture Risk in People with HIV Infection in the Probono 1 Study

Objective

To determine comparative fracture risk in HIV patients compared with uninfected controls.

Design

A randomised cross-sectional study assessing bone mineral density (BMD), fracture history and risk factors in the 2 groups.

Setting

Hospital Outpatients.

Subbbjects

222 HIV infected patients and an equal number of age-matched controls. Assessments: Fracture risk factors were assessed and biochemical, endocrine and bone markers measured. BMD was assessed at hip and spine. 10-year fracture probability (FRAX) and remaining lifetime fracture probability (RFLP) were calculated.

Main Outcome Measures

BMD, and history of fractures.

Results

Reported fractures occurred more frequently in HIV than controls, (45 vs. 16; 20.3 vs. 7%; OR=3.27; p=0.0001), and unsurprisingly in this age range, non-fragility fractures in men substantially contributed to this increase. Osteoporosis was more prevalent in patients with HIV (17.6% vs. 3.6%, p<0.0001). BMD was most reduced, and predicted fracture rates most increased, at the spine. Low BMD was associated with antiretroviral therapy (ART), low body mass index and PTH. 10-year FRAX risk was <5% for all groups. RLFP was greater in patients with HIV (OR=1.22; p=0.003) and increased with ART (2.4 vs. 1.50; OR= 1.50; p=0.03).

Conclusions

The increased fracture rate in HIV patients in our relatively youthful population is partly driven by fractures, including non-fragility fractures, in men. Nonetheless, these findings may herald a rise in osteoporotic fractures in HIV patients. An appropriate screening and management response is required to assess these risks and identify associated lifestyle factors that are also associated with other conditions such as cardiovascular disease and diabetes.