Camptothecin causes cell cycle perturbations within T-lymphoblastoid cells followed by dose dependent induction of apoptosis

We have investigated the effect of the anticancer compound, camptothecin on Jurkat T-cells, a lymphoblastoid leukemic cell-line. Exposure to low concentrations led to rapid cessation of DNA (more than 95%) and RNA (more than 75%) synthesis. Perturbations to the cell cycle were observed following exposure which caused a significant accumulation of cells within G1 (P = 0.03) with a concomitant decrease in G2/M (P = 0.025). Concentrations below 0.1 microM could inhibit DNA synthesis but not induce apoptosis. Induction of apoptosis was dose dependent and could be detected as early as 3 h post exposure. The apoptotic population appeared to be derived from G1 and S-phase cells but not G2/M, coinciding with the cell cycle compartments in which DNA and RNA polymerases function. However, direct inhibition of DNA polymerase alone was not shown to be associated the induction of apoptosis or with a decrease in susceptibility to camptothecin-induced cell death. The effects of camptothecin on Jurkat T-cells and the potential mechanisms involved are discussed in the context of observations made in other transformed cell lines.     

Nonulcerating bacterial keratitis associated with soft and rigid contact lens wear

Most patients in acute myocardial infarction (AMI) undergo emergent coronary angiography (CAG). However, when to analyze lipoprotein profiles in AMI is not clear. To determine whether lipoprotein profiles change during catheterization, we measured serum lipid and apolipoprotein concentrations in 65 patients (51 men and 14 women) before and after catheterization. Heparin was injected at 50 units/kg for CAG and 200 units/kg for percutaneous transluminal coronary angioplasty (PTCA). We found that cholesterol and triglyceride decreased by 9.4% (P < 0.001) and 53.1% (P < 0.001), respectively, after catheterization. Apolipoproteins also decreased significantly. Variables decreased two to five times more after PTCA than after CAG. Lipoprotein lipase mass was higher after PTCA (267.8 +/- 135.3 micrograms/L) than after CAG (93.3 +/- 48.4 micrograms/L; P < 0.05). In conclusion, lipoprotein profiles change during catheterization. We recommend avoiding analysis of lipoprotein profiles after emergent CAG in AMI.     

Specific interaction between tetrandrine and Quillaja saponins in promoting permeabilization of plasma membrane in human leukemic HL-60 cells

Spontaneous Ni2+ entry (leak), measured as fluorescence quench in fura-2-loaded HL-60 cells at the excitation wavelength of 360 nm, was strongly inhibited by tetrandrine (TET, 100 microM), a Ca2+ antagonist of Chinese herbal origin. Exposure of the cells for 5 min to saponins from Quillaja saponaria (QS, 30 microg/ml), surfactants well known to permeabilize the plasma membrane by complexing with cholesterol, promoted Ni2+ entry without causing fura-2 leak-out. Unexpectedly, TET caused an immediate (within 2.5 min) augmentation of QS-promoted Ni2+ entry; and a 5-min treatment with both TET and QS resulted not only in an enhanced Ni2+ entry, but also a fura-2 leak-out. Ginseng saponins (100 microg/ml) alone or together with TET did not cause such a permeabilization. Permeabilization induced by 1-3 microM digitonin, another cholesterol-complexing glycoside, could not be enhanced by TET. TET did not affect permeabilization induced by Triton X-100 (0.01%), a detergent which non-specifically disrupts the hydrophobic interaction at the plasma membrane. TET also did not enhance Ni2+ entry triggered by ionomycin (0.35 microM) or SK&F 96365 (20 microM). Further, it did not augment Ni2+ entry when the plasma membrane fluidity was modulated by changes of temperature (27-47 degrees C) or treatment with 5% ethanol. This QS-promoted Ni2+ entry could not be amplified by other lipophilic Ca2+ antagonists, such as diltiazem (100 microM) and verapamil (100 microM). The results hence indicate that TET enhanced Ni2+ entry (or permeabilization) elicited by QS treatment, but not other perturbations of the plasma membrane. We suggest that pore formation at the plasma membrane, a consequence of QS-cholesterol interaction, can be specifically enhanced by TET. Also, a comparative study of the effects of TET and its very close analogues, hernandezine and berbamine, reveals that the methoxyl group at the R2 position of TET appears to be crucial in enhancing QS-promoted Ni2+ entry.     

Cell-free V(D)J recombination

V(D)J recombination generates diversity in the immune system through the lymphoid-specific assembly of multiple gene segments into functional immunoglobulin and T-cell receptor genes. The first step in V(D)J recombination is cleavage of DNA at recombination signal sequences. Cleavage produces a blunt DNA end on each signal sequence and a hairpin end on adjacent coding gene segments, and can be reproduced in vitro by using purified RAG and RAG2 proteins. The later steps involve processing and joining of the cleaved DNA ends, and until now have been studied only in cells. Here we reconstitute the complete V(D)J recombination reaction in a cell-free system. We find that the RAG proteins are not only involved in cleavage, but are also needed in the later steps for efficient joining of coding ends. Joining is largely directed by short pieces of identical sequence in the coding flanks, but addition of human DNA ligase I results in greater diversity. Coding junctions contain short deletions as well as additions complementary to a coding flank (P nucleotides). Addition of non-templated nucleotides into coding junctions is mediated by terminal deoxyribonucleotidyl transferase. The cell-free reaction can therefore reproduce the complete set of processing events that occur in cells.     

Cephalosporin vs penicillin

Smaller individual series on the outcome of laparoscopic hernioplasty techniques have been reported. This study reports on the complications of 3,229 laparoscopic hernia repairs performed by the authors in 2,559 patients. The TAPP (transabdominal preperitoneal) technique was the most frequently performed: 1,944 (60%). The totally preperitoneal technique was performed 578 (18%) times. The IPOM (intraperitoneal onlay mesh) repair was performed 345 (11%) times. The plug-and-patch technique was used 286 (9%) times and simple closure of the hernia defect without mesh was used in 76 (2%) repairs. Overall, there were 336 (10%) complications: 17 (0.5%) major and 265 (8%) minor. There were 54 (1.6%) recurrences, with a mean follow-up of 22 months. The TAPP technique had 19 (1%) recurrences and 141 (7%) complications. There were four bowel obstructions in this subgroup from herniation of small bowel through the peritoneal closure and trocar sites. The totally preperitoneal technique had no recurrence and 60 (10%) complications. The IPOM group had 7 (2%) recurrences and 47 (14%) complications. The plug-and-patch technique had 26 (9%) recurrences and 24 (8%) complications. The simple closure of the internal ring had 2 (3%) recurrences and 10 (13%) complications. Laparoscopic hernioplasty is not without complications. Laparoscopic hernioplasty is not without complications. Training, experience, and attention to technique will prevent some of these complications.     

Contact lens use after corneal trauma

BACKGROUND: This study was performed to explore the epidemiology and visual outcomes of major anterior segment trauma cases referred to the Contact Lens Service (CLS) in a large, urban, eye trauma center at a university hospital. METHODS: All eye trauma patients seen on the CLS at The University of Illinois in Chicago for a 2-year period were identified (n = 122). A search of all eye trauma cases seen at UIC (n = 2279) during the same period yielded 757 cases with the same diagnostic codes. RESULTS: CLS cases represent 16.1% of cases with similar diagnoses and 5.35% of all cases seen during the period of study. Cases were predominantly young men (mean age, 28 years). The mean age for CLS cases (mean age, 17 years) was significantly younger. Best-corrected visual acuity was 20/100 or better in 75% of cases with a contact lens and in 68% of cases with a spectacle lens; 82% of CLS cases had iris damage versus 14.5% of non-CLS cases; 73% of CLS cases were aphakic; 105 CLS cases were given contact lenses. Mean follow-up time was 24 months. Mean wearing time was 11 hours/day. On interview, 35 of 79 cases (44%) continued to wear lenses. CONCLUSIONS: Cases referred to the CLS tended to be young men with unilateral injuries-predominantly open globes-with resultant aphakia and iris damage. Good visual acuity was achieved in most cases. Long-term success wearing contact lenses for this population was poor, suggesting that alternatives to contact lens wear should be more actively considered.     

Inhibition of G protein-coupled receptor kinase subtypes by Ca2+/calmodulin

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous betaARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gbetagamma-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 approximately 2 microM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases.