Innocuousness and intracellular distribution of PKH67: A fluorescent probe for cell proliferation assessment

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.     

Effect of temperature and photoperiod on the growth of the embryonic dogfish, Scyliorhinus canicula

Photoperiod has no effect on the growth of embryonic dogfish. Conversely, developmental rates are highly susceptible to temperature variation. Embryos maintained at 16°C grew faster and ecloded earlier than those maintained at 10°C. Increased temperature had no effect on size at eclosion.     

Cover Image: Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P‐mediated autophagy‐independent pathway (Cellular Microbiology 1/2021)

Immunofluorescence image of Plasmodium berghei sporozoites (red) in HeLa cells 1.5 hours post infection. The parasitophorous vacuole membrane (PVM) that surrounds the parasites was stained with antibodies against the PVM‐resident protein UIS4 (grey). DNA was stained with DAPI (blue). Imaging was performed on a Leica SP8 confocal microscope. For further details, readers are referred to the article by Bindschedler et al. on p. e13271 of this issue.

     

Distribution pattern of three neural calcium-binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retina

Summary Neural Ca²⁺-binding proteins (NCaPs) constitute a subfamily of 4-EF-hand proteins, and display a histological and structural dichotomy: the A-type NCaPs are selectively expressed by the retina and pineal organ and display two canonical EF-hands, whereas the B-type NCaPs are found in the entire brain and present three regular EF-hands. In this study, antisera were raised against the A-type NCaP recoverin (26 kDa) and the B-type NCaPs VILIP and NCS-1 (22 kDa). Since the sequence identity among NCaPs is high, specific polyclonal antibodies were purified by double cross-immunoaffinity chromatography; both ELISA and immunoblot analyses determined that the resulting antibodies showed selectivity ratios inferior to 1/363 for the two other related NCaPs. Besides, the anti-VILIP antibodies displayed some affinity toward neurocalcin δ, and the antirecoverin antibodies recognized a 24 kDa protein, which is most likely visinin. Thus, immunohistochemical studies on the chicken, rat and cow retina revealed that anti-recoverin antibodies recognized the vertebrate photoreceptors and a small number of mammalian bipolar cells. Anti-VILIP antibodies exclusively labelled the inner Retina, I.e. the amacrine and ganglion cells. NCS-1 was mainly present in the photoreceptor inner segments, the inner plexiform layer and the ganglion cells. NCS-1 showed the highest species disparity. The retinal localization of NCS-1 and VILIP offered an important morphological basis for the understanding of their function. Furthermore, specific antibodies against the NCaPs may enable the identification of cell populations in more complex neural tissues, such as the brain.     

Race,Menopause, Health-Related Quality of Life,and Psychological Well-Being in Obese Women

Objective: To investigate the health-related quality of life (HR-QOL) in African-American (AA) and white (W) obese women. Research Methods and Procedures: Participants were 145 obese women (80 AA and 65 W; 87 premenopausal and 58 postmenopausal) who completed the Medical Outcomes Study short form, the Brief Symptom Inventory, the Life Distress Inventory, the Satisfaction With Life Scale, and the Rosenberg Self-Esteem Scale before entering a weight-loss study. The mean age of the subjects was 46.3 ± 11.1 years and the mean body mass index was 35.2 ± 4.2 kg/m². Results: Although AA women were slightly heavier (95.3 ± 10.3 kg vs. 91.5 ± 11.6 kg, p < 0.05) and less educated (14.2 ± 3.7 years vs. 15.7 ± 3.7 years, p < 0.05) than the W women in the sample, there was no difference between the two ethnic groups in any of the reported HR-QOL variables. Menopausal status had a significant effect on HR-QOL, with premenopausal women being more distressed (p = 0.002), having more limitations in social activity (p = 0.007), and having less vitality (p < 0.001) than the postmenopausal women. This was especially true in the AA women. Discussion: These data show no difference in HR-QOL between AA and W obese women and suggest that menopausal status may have an impact on HR-QOL, especially in AA women.     

Scedosporium (Monosporium) apiospermum: multiple brain abscesses

A 32-year-old female with alcoholic cirrhosis attempted to drown herself in a pond. She remained in a coma for 57 days. After a stormy course she developed multiple brain abscesses due to Scedosporium (Monosporium) apiospermum. Despite intravenous and intrathecal amphotericin B (total dose 870 mg) the patient died 71 days after the near drowning.     

Regional Subcutaneous‐Fat Loss Induced by Caloric Restriction in Obese Women

Objective: With anthropometric models using skinfolds and circumferences, we studied changes in the percentage of subcutaneous fat in the total cross‐sectional area (SF%) at four body sites in obese women, before and after weight loss induced by 6 months of caloric restriction. Research Methods and Procedures: In 61 obese women [31 African Americans and 30 whites; ages, 24 to 68 years; body mass index (BMI), ≥28kg/m²], we measured SF% at the midpoint of the upper arm and thigh and the waistline and hipline, and we measured the percentage of total body fat by DXA before (Obs#1) and after (Obs#2) a 6‐month nonintervention control period and then after 6 months on a 1200 kcal/d diet (Obs#3). Results: The mean body weight and BMI increased (1.8 kg and 0.61 kg/m²; p = 0.0001), but there were no significant changes in any other body composition measurements from Obs#1 to Obs#2. The means of Obs#3 for weight and BMI decreased by 11%, and the percentage of total body fat decreased by 13% of Obs#2 mean values (p = 0.0001). The mean SF% at each site decreased 7.6% to 18.0% of the Obs#2 mean values (p < 0.001). The SF% decreases were greater at mid‐arm and mid‐thigh than in the cross‐sectional regions at the waistline and hipline (p = 0.05). There was no interaction between age or ethnicity (p > 0.2). Conclusions: In obese women, caloric restriction alone reduces anthropometrically measured subcutaneous fat proportionally more in peripheral than in central regions.     

Vesicular preparation of a highly coupled ATPase-ATP synthase complex from pig heart mitochondria

1. A method is described to prepare an ATPase-ATP synthase complex from pig heart mitochondria exhibiting a very high ATP-32Pi exchange activity (1.6 mumol/min per mag protein in optimal conditions). 2. The preparation is virtually devoid of nucleoside diphosphokinase and adenylate kinase activities. 3. Freeze-fracture studies show that the ATPase-ATP synthase complex is integrated in lipid vesicles of 400-600 A in diameter. 4. It contains the endogenous natural proteic inhibitor which seems to behave as a coupling factor. 5. The rate of ATP hydrolysis catalyzed by the ATPase-ATP synthase complex is competitively inhibited by ADP, while the presence of ADP increases the initial rate of 32Pi incorporation into ATP. 6. The 32Pi incorporation into ATP can occur at a rate almost equal to that of nucleoside triphosphate (NTP) hydrolysis provided that the rate of NTP hydrolysis is kept low and that the ADP concentration is high enough. In these conditions, a very high coupling between NTP hydrolysis and ATP synthesis can be demonstrated.