On-line identification of unstable iridoids from Jamesbrittenia fodina by HPLC-MS and HPLC-NMR

HPLC-UV-MS analysis of the methanol extract of Jamesbrittenia fodina (Wild) O. M. Hilliard (Scrophulariaceae) revealed the presence of different iridoid cinnamic esters; however, isolation of these constituents was prevented by instability problems. HPLC-UV-MS and HPLC-NMR analysis of the mixtures obtained after a tentative isolation indicated that, in the first instance, instability was due to a light-induced cis/trans isomerisation of the cinnamic moieties. Further investigation of related compounds showed an additional instability problem linked to other chemical transformations. A detailed HPLC-NMR-MS study of these fractions demonstrated that the modifications occurred on the rhamnose moiety of these iridoids. It could be concluded that the second type of instability was attributable to transesterification of the cinnamic moiety on the rhamnose unit. The recording of stop-flow HPLC-NMR spectra for specific HPLC peaks permitted the direct monitoring of these transformations. Based on these on-line data, six new unstable aucubin derivatives were efficiently characterised.     

Meiosis: cell-cycle controls shuffle and deal

Meiosis is the type of cell division that gives rise to eggs and sperm. Errors in the execution of this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Here, we review recent findings on how cell-cycle controls ensure the coordination of meiotic events, with a particular focus on the segregation of chromosomes.     

Actomyosin cross-linking by caldesmon in non-muscle cells

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.     

Risk Factors for Inadequate TB Case Finding in Rural Western Kenya: A Comparison of Actively and Passively Identified TB Patients

Background

The findings of a prevalence survey conducted in western Kenya, in a population with 14.9% HIV prevalence suggested inadequate case finding. We found a high burden of infectious and largely undiagnosed pulmonary tuberculosis (PTB), that a quarter of the prevalent cases had not yet sought care, and a low case detection rate.

Objective and methods

We aimed to identify factors associated with inadequate case finding among adults with PTB in this population by comparing characteristics of 194 PTB patients diagnosed in a health facility after self-report, i.e., through passive case detection, with 88 patients identified through active case detection during the prevalence survey. We examined associations between method of case detection and patient characteristics, including HIV-status, socio-demographic variables and disease severity in univariable and multivariable logistic regression analyses.

Findings

HIV-infection was associated with faster passive case detection in univariable analysis (crude OR 3.5, 95% confidence interval (CI) 2.0–5.9), but in multivariable logistic regression this was largely explained by the presence of cough, illness and clinically diagnosed smear-negative TB (adjusted OR (aOR) HIV 1.8, 95% CI 0.85–3.7). Among the HIV-uninfected passive case detection was less successful in older patients aOR 0.76, 95%CI 0.60–0.97 per 10 years increase), and women (aOR 0.27, 95%CI 0.10–0.73). Reported current or past alcohol use reduced passive case detection in both groups (0.42, 95% CI 0.23–0.79). Among smear-positive patients median durations of cough were 4.0 and 6.9 months in HIV-infected and uninfected patients, respectively.

Conclusion

HIV-uninfected patients with infectious TB who were older, female, relatively less ill, or had a cough of a shorter duration were less likely found through passive case detection. In addition to intensified case finding in HIV-infected persons, increasing the suspicion of TB among HIV-uninfected women and the elderly are needed to improve TB case detection in Kenya.     

Histomathematical analysis of clinical specimens: challenges and progress.

Proteomic analysis of clinical tissue specimens is a difficult undertaking. Described here is a multiplex study of protein expression levels in histological sections of human prostate that addresses many of the associated challenges. Whole-mount sections from 10 prostatectomy specimens were studied using 15 antibodies, immunohistochemical staining, digital imaging, and mathematical analysis of the data sets. The approach was successful in stratifying cell lineages present in the samples based on proteomic patterns, including differentiating normal epithelium from cancer. This strategy likely will be a useful method for extending the number of proteins that can be analyzed in clinical cancer specimens using currently available laboratory techniques.     

Leptin does not directly affect CNS serotonin neurons to influence appetite

Serotonin (5-HT) and leptin play important roles in the modulation of energy balance. Here we investigated mechanisms by which leptin might interact with CNS 5-HT pathways to influence appetite. Although some leptin receptor (LepRb) neurons lie close to 5-HT neurons in the dorsal raphe (DR), 5-HT neurons do not express LepRb. Indeed, while leptin hyperpolarizes some non-5-HT DR neurons, leptin does not alter the activity of DR 5-HT neurons. Furthermore, 5-HT depletion does not impair the anorectic effects of leptin. The serotonin transporter-cre allele (Sert(cre)) is expressed in 5-HT (and developmentally in some non-5-HT) neurons. While Sert(cre) promotes LepRb excision in a few LepRb neurons in the hypothalamus, it is not active in DR LepRb neurons, and neuron-specific Sert(cre)-mediated LepRb inactivation in mice does not alter body weight or adiposity. Thus, leptin does not directly influence 5-HT neurons and does not meaningfully modulate important appetite-related determinants via 5-HT neuron function.     

Food resource allocation patterns in lactating females in a long-term selection experiment for litter size in mice

Resource allocation patterns, as quantified by residual food intake (RFI), and the consequences for offspring development were investigated during lactation in 96 females of a mouse line selected for 104 generations for high litter size at birth (S-line) and in 87 females of a non-selected control line (C-line). Litters of 45 C-line dams (Cs) and 48 S-line dams (Ss) were standardised (s) at birth; other dams (ns) supported total number of pups born (Cns and Sns, respectively). RFI during lactation was significantly lower in Sns-dams than in C-line dams and Sns-dams. After weaning Sns-dams seemed to be able to restore the negative resource situation. Sns-pups were about 25% less mature than Cns-pups at all times. Maturity was similar for Cs- and Ss-pups from 2 d in lactation on, and about 18% and 53% higher than Cns- and Sns-pups. The pre-weaning mortality rate was significantly higher in Sns-litters (35.6 ± 2.76) than in Cns-litters (4.95 ± 2.23). The results suggest that S-line dams allocated considerably more resources to maintenance of offspring than C-line dams. This was insufficient to provide the offspring with an adequate amount of resources, resulting in reduced pup development and increased pre-weaning mortality rates.     

Nucleotide binding to myosin in calcium activated muscle

The nucleotides bound to calcium-activated muscle fibres or myofibrils were separated and estimated. Most of the bound nucleotide was found to be ADP, as in relaxed muscle. The maximum ADP binding was not significantly altered by activation but the dissociation constant of the bound ADP was slightly reduced. These results are discussed in terms of the possibility of formation of a ternary ADP—myosin—actin complex.     

GENETIC VARIABILITY AND POTENTIAL SOURCES OF GRATELOUPIA DORYPHORA (HALYMENIACEAE,RHODOPHYTA), AN INVASIVE SPECIES IN RHODE ISLAND WATERS (USA)1

Grateloupia doryphora (Montagne) M.Howe is an invasive foliose alga that was reported for the first time in Rhode Island, USA in 1997. The population has since increased in size and expanded in range. In this study, the genetic variation and potential sources of the Rhode Island G. doryphora population were examined using three types of molecular markers: randomly amplified polymorphic DNA (RAPD), nuclear internal transcribed spacer (ITS) sequences, and mitochondrial cox2–cox3 intergenic spacer (COX) sequences. No variation was detected in ITS or COX sequences among Rhode Island G. doryphora individuals. RAPDs, however, did reveal genetic variation, although banding patterns were similar, with RAPD genetic distances between individuals ranging from 0.00 to 0.17. The low level of genetic diversity observed within the Rhode Island population may be due to a small founder population or a founder population derived from a genetically uniform source. To identify possible sources of the Rhode Island invasion, individuals from nine geographically diverse populations of foliose Grateloupia were compared. Phylogenetic trees inferred from RAPD distances and ITS and COX sequences had similar topologies; thus there was phylogenetic congruence among these independent loci. The Rhode Island G. doryphora specimens were genetically similar to specimens from G. doryphora populations located in Portsmouth, England; Tholen Island, The Netherlands; and Brittany and Hérault, France. Interestingly, the G. doryphora population in each of these locations is itself due to an introduction event within the past 40 years.     

The denatured state of N-PGK is compact and predominantly disordered

The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I_b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I_k) and denatured (D)]. The transition to I_b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I_b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I_b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I_b indicate that it forms from the fully denatured state within 100 μs, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I_b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.