Variation in cell-substratum adhesion in relation to cell cycle phases

The quantification of focal adhesion sites offers an assessable method of measuring cell-substrate adhesion. Such measurement can be hindered by intra-sample variation that may be cell cycle derived. A combination of autoradiography and immunolabelling techniques, for scanning electron microscopy (SEM), were utilised simultaneously to identify both S-phase cells and their focal adhesion sites. Electron-energy 'sectioning' of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy 'plane' image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labelled focal adhesions sites on the same cell. The focal adhesion densities were calculated both for identified S-phase cells and the remaining non-S-phase cells present. The results indicated that the cell cycle phase was a significant factor in determining the density of focal adhesions, with non-S-phase cells showing a larger adhesion density than S-phase cells. Focal adhesion morphology was also seen to correspond to cell cycle phase; with 'dot' adhesions being more prevalent on smaller non-S-phase and the mature 'dash' type on larger S-phase cells. This study demonstrated that when quantitation of focal adhesion sites is required, it is necessary to consider the influence of cell cycle phases on any data collected.     

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.     

β-Glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are β-glucuronidase (β-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that β-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas β-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, β-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that β-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed. This study was funded by the University of the Basque Country through a grant to Consolidated Research Groups. U.I. is a recipient of a pre-doctoral fellowship from the Basque Government.     

High Transmitter CD4+ T-Cell Count Shortly after the Time of Transmission in a Study of African Serodiscordant Couples

Background

2013 WHO guidelines recommend starting ART at CD4+ T-cell counts ≤500 cells/μL. We present the T-cell counts from adult Africans with HIV shortly following transmission to their sexual partners.

Methods

HIV-discordant couples in Zambia, Uganda and Rwanda were followed prospectively and received couples counseling and condoms. HIV uninfected partners were tested for HIV at least quarterly and HIV-infected partners received HIV care and referral for ART per national guidelines. Upon diagnosis of incident HIV infection in the previously HIV-uninfected partner, a blood sample was collected from both partners to measure CD4+ T-cells and perform viral linkage. The estimated date of infection (EDI) of the incident case was calculated based on testing history. EDI was unknown for suspected transmitting partners.

Results

From 2006–2011, 4,705 HIV-discordant couples were enrolled in this cohort, and 443 cases of incident HIV infection were documented. Virus linkage analysis was performed in 374 transmission pairs, and 273 (73%) transmissions were linked genetically. CD4 counts in the transmitting partner were measured a median of 56 days after EDI (mean:90.5, min:10, max:396). The median CD4 count was 339 cells/μl (mean:386.4, min:15, max:1,434), and the proportion of partners with a CD4+ T-cell count above 500/μl was 25% (95% CI:21, 31).

Conclusions

In our cohort of discordant couples, 73% of HIV transmissions occurred within the relationship, and the transmitter CD4+ T cell count shortly after the transmission event was frequently higher than the WHO 2013 ART-initiation guidelines.     

Formation and function of cytoplasmic annulate lamellae in the eggs ofPomatoceros triqueter L.

Summary During an ultrastructural study of the eggs of the serpulid wormPomatoceros triqueter L., annulate lamellae were frequently encountered in the cytoplasm. In particular, some observations indicated that they originate by successive outfoldings of the nuclear envelope. Consequently, annulate lamellae must consist of alternating layers of nuclear and cytoplasmic material, each layer being separated by part of the nuclear envelope. It was observed that there was a similarity between nuclear and inter-annulate lamellar material. Moreover tritiated thymidine was shown to be present in the stacks. It is inferred that this system might well function as an efficient means of transporting nuclear material into the cytoplasm. The authors wish to thank Messrs. P. C. Lloyd, P. Henley and D. Williams for technical assistance.     

Plasma membrane transport of 2-ketoisocaproate by rat hepatocytes in primary culture

Others have shown that the branched chain 2-keto acids are generated in muscle, released into the bloodstream, and then removed by the liver where further catabolism occurs. The present investigation describes the plasma membrane transport systems for these metabolites in cultured rat hepatocytes. One of these systems in Na+-dependent, concentrates the 2-keto acids against a gradient, and is inhibited by pyruvate. The second process is Na+-independent, is less concentrative, and may be composed of two distinct systems as suggested by pyruvate inhibition studies. None of these systems accept neutral amino acids. For the transport of 2-ketoisocaproate, the Na+-dependent system exhibits a Km value of about 5 mM, whereas the corresponding value for the Na+-independent agency is 60 microM. The activity of the Na+-dependent system is moderately increased by insulin treatment of the cells, while neither agency is stimulated by glucagon, dexamethasone, or the combination of these two hormones. Hepatocytes from diabetic rats show enhanced transport by the Na+-dependent system and incubation of cultured hepatocytes for 24 h in the absence of 2-keto acids results in a 3-fold stimulation of the Na+-dependent system, but has no effect on the rate of Na+-independent transport. These results demonstrate the existence of at least two saturable transport systems for the branched chain 2-keto acids in the rat hepatocyte and the ability of the Na+-dependent system to respond to the extracellular environment.