T cell receptor/CD3 complex internalization following activation of a cytolytic T cell clone: evidence for a protein kinase C-independent staurosporine-sensitive step.

The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti-TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA). After binding of the anti-TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti-TcR mAb. PMA-induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC-depleted cells or in the presence of the PKC inhibitor staurosporine. Anti-TcR mAb-induced internalization was maintained in PKC-depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti-TcR mAb Fab fragment is non-stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC-depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA-induced phosphorylation of gamma and epsilon subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross-linking of the TcR via plastic-coated anti-TcR mAb led to phosphorylation of CD3 gamma and epsilon and also of zeta, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti-TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.     

Sex differences in rapid nonclassical action of 17β‐oestradiol on intracellular signalling and oestrogen receptor α expression in basal forebrain cholinergic neurones in mouse

Rapid nonclassical effects of 17β‐oestradiol (E₂) on intracellular signalling have been identified in the basal forebrain, although the extent to which these actions may be different in males and females is unknown. Previous work has shown that E₂ rapidly phosphorylates cAMP responsive element binding protein (CREB) via ΕRα in female cholinergic neurones. Using this indicator, the present study examined whether nonclassical actions of E₂ occur in a sexually dimorphic manner within basal forebrain cholinergic neurones in mice. In addition, we investigated the expression and intracellular distribution of oestrogen receptor (ΕR)α in cholinergic neurones in female and male mice. Animals were gonadectomised and treated 2 weeks later with E₂. The number of CREB‐expressing cholinergic neurones was not altered in any of the brain regions after E₂ treatment in both males and females. However, E₂ treatment rapidly (< 15 minutes) increased (P < 0.05) the number of cholinergic neurones expressing phosphorylated CREB (pCREB) in the substantia innominata and medial septum but not in the striatum in female mice. By contrast, E₂ did not change pCREB expression in cholinergic neurones in male mice at any time point (15 minutes, 1 hour, 4 hours), irrespective of the neuroanatomical location. We also observed that, in females, more cholinergic neurones expressed nuclear ΕRα in all regions, whereas males showed more cholinergic neurones with cytoplasmic or both nuclear and cytoplasmic expression of ΕRα. Taken together, these results demonstrate a marked sex difference in the E₂‐induced nonclassical effect and intracellular distribution of ΕRα in basal forebrain cholinergic neurones in vivo.     

Availability of donated oocytes from an ambulatory sterilization program

An oocyte donor program was established at the Women's Medical Pavilion, Dobbs Ferry, New York, in 1987 for women lacking normal ovarian function. The oocytes were donated voluntarily in stimulated cycles by women undergoing laparoscopic sterilization. If the donors agreed to use ovulation-induction agents and be monitored, they were compensated for their time and inconvenience. Between Nov 15, 1987, and Feb 15, 1988, 194 laparoscopic sterilizations were performed. Only 41 women (21%) met certain of the eligibility criteria. Of the remaining group, only five (12%) agreed to participate. The preliminary results suggest that tubal ligation patients are reluctant to donate oocytes despite enthusiastic counseling, the relatively small risks involved and offers of financial compensation.     

Direct trocar insertion vs. Verres needle use for laparoscopic sterilization

A randomized, prospective trial was designed to compare direct trocar insertion with prior peritoneal insufflation with a Verres needle for laparoscopic tubal sterilization. Direct trocar insertion resulted in fewer instrument insertions (21.8% vs. 7.8%) and use of smaller volumes of CO2 (2.67 vs. 2.32 L). Direct trocar use resulted in a decrease in operating time from 9 minutes, 40 seconds in the needle group to 7 minutes, 30 seconds in the trocar group. Minor omental injuries occurred in a small percentage of each group, while serious complications occurred once in each group.     

Use of glycosylated hemoglobin as a screen for macrosomia in gestational diabetes

Glycosylated hemoglobin and blood sugar levels in the fasting state and two hours after oral 100 g glucose load were measured in 180 patients. Glycosylated hemoglobin was measured by cation exchange column chromatography, and blood sugar was measured by hexokinase reaction. Patients with an elevated postprandial and/or fasting blood sugar level (positive screen) subsequently underwent three-hour glucose tolerance test. The mean value of glycosylated hemoglobin in patients with a negative screen and normal hemoglobin was 6.17 +/- 0.61%; and the value for glycosylated hemoglobin in patients with class A diabetes and normal hemoglobin electrophoresis was 6.85 +/- 0.73% (P less than .001). A glycosylated hemoglobin value greater than 6.78 (mean + 1 SD) was considered elevated. Glycosylated hemoglobin values were elevated in 21 of 33 patients with gestational diabetes and in 27 of 147 patients with normal blood sugar levels. The sensitivity and specificity of glycosylated hemoglobin for the diagnosis of gestational diabetes were 63.6 and 81.6%, respectively. Fifty percent of patients with an initially elevated glycosylated hemoglobin value delivered macrosomic infants, whereas no patient with a normal glycosylated hemoglobin value had a macrosomic infant. An elevated glycosylated hemoglobin value may alert the obstetrician of a potentially elevated mean blood sugar level and may warrant aggressive management of gestational diabetes.     

Performance of a new, low-volume, high-surface area aqueous shunt in normal rabbit eyes

PURPOSE: This experimental study was conducted to report perfusion characteristics of small diameter, cylindrical aqueous shunts in normal rabbit eyes and to test the hypothesis that decreasing bleb diameter would decrease capsular fibrosis, as evidenced by a thinner capsule forming around the implant. These two properties increase hydraulic conductivity of the fibrous membrane forming around the device, resulting in a more effective filtering shunt. METHODS: Cylindrical latex tubes with the distal portion of the sidewall removed were implanted under the conjunctiva. The proximal, intact end of tubing was inserted into the anterior chamber and ligated to prevent hypotony. The ligature was released after 1 week to inflate the bleb. Animals were again anesthetized at 6 or 12 weeks after ligature release and in vivo perfusion experiments conducted using a miniperfusion system and a water manometer. Perfusion of the implant with latex microspheres was performed before the animals were killed. Capsule diameters were measured in situ using calipers under a dissecting microscope after excision of orbital tissues and before fixation for histologic study. Membrane hydraulic conductivity (outflow per unit membrane area at unitary pressure gradient, microL/min/mm2/mmHg) was calculated using data obtained during perfusion experiments and compared with results of other studies. Capsule thickness was measured histologically. RESULTS: Cylindrical filtration membranes with thin (15-20 microm) capsules formed around latex implants. Bleb diameters consistently measured 1 mm at all points along their length before formaldehyde fixation. Hydraulic conductivity was measured and found to be eight times higher than that reported for capsules around conventional implants. Decreased diameter (16:1 for Baerveldt and 13:1 for Molteno implants) produced a proportional decrease in surface tension on the wall. This resulted in decreased capsule thickness, from 222 microm for Baerveldt implants in rabbit eyes and from 150 microm for Molteno implants in monkey eyes to less than 20 microm for the shunting device studied here. CONCLUSION: As a consequence of Laplace's law, reduction in bleb diameter reduces surface tension on the bleb, reducing capsular fibrosis and consequently capsule thickness, thus increasing hydraulic conductivity. Increased hydraulic conductivity increases the effectiveness of the filtering surface. Cylindrical geometry allows reduced bleb volume yet maintains total surface area that is proportional to the length of the implant, which is unlimited and customizable for each eye by simply cutting the length of implant needed.