Cutaneous leishmaniasis responds to daylight-activated photodynamic therapy: proof of concept for a novel self-administered therapeutic modality.

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in Israel, with hundreds of new cases reported in recent years. Photodynamic therapy (PDT) is highly effective for treatment of CL, but requires equipment available only at specialized centres. Daylight-activated PDT (DA-PDT) abolishes the need for artificial light sources and allows the patient to administer the treatment with no professional assistance. OBJECTIVES: The objective of this single-centre, open study was to establish proof of concept for the efficacy of DA-PDT in the treatment of CL using clinical, microbiological and molecular clearance as outcome measures. METHODS: Thirty-one patients with CL (11 Leishmania major and 20 Leishmania tropica) underwent DA-PDT. Fourteen patients were treated in the hospital garden under professional supervision and 17 patients underwent DA-PDT as a self-administered treatment modality at home. Following application of a thick layer of 16% methyl aminolaevulinate and 30-min occlusion, the lesions were exposed to daylight for 2·5 h. Treatment sessions were repeated at weekly intervals until clinical and microbiological cure. Control lesions were either treated with cryotherapy or left untreated. RESULTS: The overall cure rate for DA-PDT was 89% (intention-to-treat cure rate 77%); this was 86% for the hospital-based treatment group and 92% for the self-administered group. CONCLUSIONS: DA-PDT proved to be effective in the treatment of CL caused by L. major and L. tropica. More patients were treated according to a self-administered protocol, suggesting that DA-PDT can be adopted even in technologically deprived countries where the majority of Leishmania infections are encountered.

Anti-TNP antibody localization of the reactive lysine residues in myosin.

Myosin contains reactive lysine residues which are trinitrophenylated by 2,4,6-trinitrobenzene sulfonate much faster than the rest of the lysines. Here we find the location of these residues in the primary and spatial structure of myosin with the help of an anti-trinitrophenyl antibody. This antibody was raised against trinitrophenyl hemocyanin in rabbits. It reacted with trinitrophenylated myosin, and with some of the tryptic fragments of trinitrophenylated myosin. By analyzing the reaction with Western blots, it was found that the antibody preferentially reacts with the 27 kDa N-terminal fragment of the myosin head, and more weakly with the light meromyosin region of the myosin rod. The 27 kDa fragment contains the most reactive lysine residue, while the intermediate lysine residue is located in the light meromyosin region. The locations of the epitopes of the antibody were visualized on electron microscope images of rotary-shadowed trinitrophenylated myosin-antibody complexes. The distances of the epitopes to the head-rod junction of myosin were measured as 13 and 113 nm for the epitope on the head (reactive lysine residue) and for that on the rod (intermediary reactive lysine residue), respectively.

Electroejaculation before chemotherapy in adolescents and young men with cancer.

OBJECTIVE: To evaluate the outcome of repeated electroejaculation for obtaining semen from adolescents and young men before initiation of anticancer therapies. DESIGN: Retrospective clinical study. SETTING: Bikur Cholim Hospital, Jerusalem, Israel. PATIENT(S): Six young male patients (average age, 18+/-3 years) with diagnosed cancer who underwent 12 procedures of electroejaculation before chemotherapy. INTERVENTION(S): Transrectal electroejaculation. Semen was cryopreserved in small aliquots. MAIN OUTCOME MEASURE(S): Semen analysis. RESULT(S): In all patients, semen was obtained by electroejaculation. Sperm count and motility were relatively low; mean values were 16 x 10(6) (range, 0--45 x 10(6)) and 14% (range, 0--53%) respectively. CONCLUSION(S): If necessary, electroejaculation can be performed in adolescents, and sperm may be obtained by repeated treatments over a short period.

Electroejaculation in combination with intracytoplasmic sperm injection in patients with psychogenic anejaculation results in lower fertilization rates.

OBJECTIVE: To evaluate the outcome of intracytoplasmic sperm injection (ICSI) with sperm obtained by electroejaculation in men with psychogenic anejaculation. DESIGN: Retrospective clinical study. SETTING: In Vitro Fertilization Unit, Bikur Cholim Hospital, Jerusalem, Israel. PATIENT(S): Seven men with psychogenic anejaculation who underwent 16 sessions of electroejaculation in combination with ICSI. INTERVENTION(S): Electroejaculation, ICSI. MAIN OUTCOME MEASURE(S): Semen analysis, ICSI, fertilization rates. RESULT(S): All patients had poor sperm motility. One hundred forty-seven oocytes were injected, with a fertilization rate of 27% (39/142). One ongoing pregnancy was achieved. CONCLUSION(S): Sperm obtained by electroejaculation have low motility and reduced fertilization potential. Nevertheless, ICSI should be offered to improve the possibility of successful pregnancy.

Nocturnal sperm emission in men with psychogenic anejaculation.

OBJECTIVE: To evaluate sperm characteristics and fertilization potential in sperm obtained from nocturnal emission in men with psychogenic anejaculation. DESIGN: Retrospective study. SETTING: In Vitro Fertilization Unit, Bikur Cholim Hospital, Jerusalem, Israel. PATIENT(S): Six men with psychogenic anejaculation. INTERVENTION(S): Nocturnal emission, electroejaculation, sperm cryopreservation, and assisted reproduction technologies. MAIN OUTCOME MEASURE(S): Semen analysis, intracytoplasmic sperm injection (ICSI), fertilization rates. RESULT(S): In four patients, the concentration and motility of sperm obtained from freeze-thawed nocturnal emission were decreased compared with sperm from electroejaculation. Fertilization rates after ICSI using the nocturnal emission sperm were relatively low (45%). One clinical pregnancy was achieved after intrauterine insemination. CONCLUSION(S): The quality of sperm from nocturnal emissions is variable, but it can be used in assisted reproduction procedures to avoid aggressive procedures such as electroejaculation or testis biopsy.

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction.

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.

Expression of glucose transporter and glucose uptake in human oocytes and preimplantation embryos.

The expression of glucose transporters 1, 2, 3 and 4 was evaluated in human oocytes and polyploid preimplantation embryos. Only glucose transporter 1 (GLUT-1) isoform was detected in oocytes and in 2-12-cell stage embryos. Glucose uptake was markedly increased in embryos as compared to oocytes (19.7 +/- 3.4 pmol/min/embryo and 2.3 +/- 0.3 pmol/min/oocyte), and GLUT-1 was inhibited by cytochalasin B. These results suggest that, although GLUT-1 is expressed in human oocytes and throughout preimplantation development, its function in mediating the rise in glucose uptake is triggered following fertilization.

Antisperm antibodies in men with psychogenic anejaculation.

Antisperm autoantibodies were determined in 16 men suffering from psychogenic anejaculation who underwent assisted reproduction treatments. Blood and semen samples were collected after transrectal electroejaculation and antisperm antibodies in serum and on the surface of motile spermatozoa were measured using the direct and indirect immunobead binding test. Five men (31%) were found positive for antisperm antibodies. The majority of antibodies were directed against the sperm heads. Surface antibodies were mainly IgA isotype whereas serum antibodies were IgG isotype. These results suggest that psychogenic anejaculation might be associated with increased incidence of antisperm autoimmunity.

Effect of mild heat treatment on actin and nucleotide binding of myosin subfragment 1.

Chymotryptic subfragment 1 (S-1) prepared from rabbit skeletal myosin has lost its ATPase activity upon incubation at 35 degrees C for 3 h. The loss in ATPase activity was accompanied by the perturbation of the structure of the 50K domain as indicated by a dramatic increase in the tryptic susceptibility of this domain without any change in the susceptibility of the other domains of S-1. The perturbation starts at the C-terminal region of the domain as suggested by the appearance of a 29K intermediate protein band in the tryptic peptide pattern of the heat-treated S-1. The heat-treated molecule essentially retained its actin and polyphosphate binding ability, and the actin binding was still sensitive to the presence of ATP or pyrophosphate. However, as opposed to native S-1, in heat-treated S-1 the addition of ATP does not induce an increase in tryptophan fluorescence, and, in the case of the treated species, the fluorescence of 1,N6-ethenoadenosine 5'-diphosphate added to the mixture is quenchable by acrylamide. This latter observation suggests that the binding of the adenine ring of the nucleotide has been altered following the heat treatment. The results indicate that the actin and polyphosphate binding sites of S-1 are distinct and that they are relatively independent of the adenine ring binding site.

Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosin.

Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin--27 kDa, 50 kDa and 20 kDa--aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1-23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the 'anti-27 K' and 'anti-50 K' epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the 'anti-50 K' to S-1 decreased upon actin binding, while that of the 'anti-27 K' was not affected by binding of S-1 to F-actin. The 'anti-50 K' antibody inhibited the K+ (EDTA) and the actin-activated ATPase activity of S-1, while the 'anti-27 K' had no effect. The results indicate that either the epitope of the 'anti-50 K' is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.

The use of ICSI with fresh and cryopreserved electroejaculates from psychogenic anejaculatory men.

BACKGROUND: Electroejaculation has become an accepted form of semen procurement in men suffering from anejaculation. However, sperm in these ejaculates often exhibit low motility. In such cases, ICSI is offered to improve the possibility of successful pregnancy. Here we evaluate the fertilizing potential, using ICSI, of fresh and cryopreserved sperm obtained by transrectal electroejaculation from patients with psychogenic anejaculation. METHODS: A total of 25 men suffering from psychogenic anejaculation underwent 37 sessions of electroejaculation in combination with ICSI. In 17 patients fresh sperm (29 cycles, group I) was used, and in the other eight patients cryopreserved sperm (10 cycles, group II) was used. RESULTS: A total of 155 oocytes were injected with fresh sperm with a fertilization rate of 55% (85/155). The pregnancy rate was 10% (3/29) per cycle. A total of 94 oocytes were injected with frozen-thawed sperm with a fertilization rate of 50% (47/94). The pregnancy rate was 40% (4/10) per cycle. CONCLUSIONS: The fertilization and pregnancy rates with cryopreserved sperm from electroejaculation are at least as good as those of freshly obtained sperm. Therefore, when motile sperm is found in the thawed ejaculate, additional electroejaculation can be avoided.

Substrate autoregulation of glucose transport: hexose 6-phosphate mediates the cellular distribution of glucose transporters.

Exposure of rat skeletal muscle and skeletal muscle cell lines to high glucose levels results in a time- and dose-dependent reduction of the rate of hexose uptake, paralleled by a reduction in the plasma membrane density of glucose transporters. The mechanism of this process was investigated in cultured L8 myocytes. Low concentrations (0.5-2.0 mmol/l) of deoxyglucose mimicked the downregulatory action of 20 mmol/l glucose both regarding the time-course and magnitude of the effect, but in an irreversible manner. A dose-dependent relationship between intracellular accumulation of deoxyglucose 6-phosphate and the magnitude of the downregulatory response was observed. Depletion of intracellular deoxyglucose 6-phosphate restored the rate of hexose transport to the control level. The reduction of hexose transport activity by deoxyglucose occurred independently of ATP depletion which by itself produced the opposite effect. The effects of deoxyglucose and high glucose on hexose transport were associated with reduced transport maximal velocity and GLUT1 transporter abundance in the plasma membranes of myocytes, as assessed by cell surface biotinylation. The reduction of myocyte GLUT1 mRNA content, observed after exposure to high glucose, did not accompany the transport down regulatory action of deoxyglucose. We suggest that hexose 6-phosphate is the mediator of the downregulatory signal for subcellular redistribution of GLUT1 in L8 myocytes. The signal responsible for reducing the GLUT1 mRNA level may be related to glucose metabolites downstream of the hexokinase reaction.