Expression of the alpha, beta II, and gamma protein kinase C isozymes in the baculovirus-insect cell expression system. Purification and characterization of the individual isoforms

Detailed in vitro comparisons of the biochemical characteristics of three protein kinase C isozymes were performed. As an alternative to earlier uncertain separation methods and expression schemes, highly purified and genetically distinct protein kinase C enzymes were produced using the baculovirus expression system. The baculovirus expression system yielded approximately 200-300 micrograms of the purified isozyme from 3 x 10(8) (100 ml of culture medium) baculovirus-infected insect cells. Biochemical characterization of the expressed isozymes indicated that the three isozymes had virtually indistinguishable Ca2+, Mg2+, and ATP dependencies. However, in certain critical functional characteristics such as phosphatidylserine dependencies, phospholipid and substrate preferences, and arachidonic acid activation, the gamma isozyme exhibited distinctive properties when compared with both the alpha and beta II subtypes. In addition, the activity of the beta II subtype was more dependent upon diacylglycerol or phorbol esters for activation than either the alpha or gamma isoforms. The alpha isozyme, unlike the beta II and gamma forms, was totally dependent on Ca2+ for activation in the presence of free arachidonic acid. These studies provide definitive characterizations of the pure isoforms; many of the findings were consistent with earlier enzymatic observations using hydroxyapatite-purified isoforms. Thus, the distinctive biochemical properties of the protein kinase C isozymes are consistent with the hypothesis that each isoform may have distinct roles in signal transduction processes.     

ASHG activities relative to education. Heredity and adoption: a survey of state adoption agencies.

A subcommittee of The Social Issues Committee of The American Society of Human Genetics (ASHG), aware of the growing interest in the implications of inheritance and adoption, in 1987 began surveying the 50 states' and Washington DC's public adoption agencies regarding this issue, and it completed the survey in 1988. In 1987, two surveys obtained data on each state's legal requirements for obtaining genetic information and on what each public adoption agency collected as genetic history. These results were presented in a poster at the 1987 ASHG annual meeting in San Diego. In 1988, a questionnaire was sent to the same agencies to elicit opinions as to whether adoption agencies should systematically collect genetic information to share with the adoptive family and adoptee, whether legislation should be mandated to collect such information, and whether genetic education programs should be developed and implemented for adoption-agency staff. On the basis of responses to the 1988 questionnaire, it was concluded that there is an interest in developing a uniform set of genetic information which should be part of a child's adoption record and that there is a need for genetic education programs for adoption workers. Responses to the desirability of mandating legislation for this purpose were less consistent.     

MICROSPOROGENESIS IN PEANUT (ARACHIS HYPOGAEA)

The stage of pollen development at the time of anther culture is an important factor in the production of haploids. The objectives of the current study were to develop a staining procedure for peanut (Arachis hypogaea L., ssp. hypogaea) microspores, to describe and document the stages of microsporogenesis in peanut, and to confirm a previous report concerning correlations of peanut floral bud shape with stage of microspore development. A staining procedure using propionic carmine provided adequate staining of pollen mother cells, microspores, and pollen. Pollen mother cells and microspores could easily be differentiated by their size and cell wall structure. Plants grown in a controlled environment were found to have highly synchronized microspore development, both within an anther and among anthers contained in the same bud. In addition, floral bud shape was confirmed as a reliable indicator of anther stage in peanuts.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Use of microspheres in measurement of regional blood flows during +GZ stress

The use of the radiolabeled microsphere technique for the study of the effects of +GZ acceleration on regional blood flow is examined. A theoretical analysis of the limits of this technique in a high acceleration environment is presented. Chronically implanted, electromagnetic, aortic flow probes were used to determine the relationship between aortic blood flow velocity and +GZ acceleration in conscious adult miniature swine. It was found that conscious straining adult miniature swine, with the assistance of an inflated anti-G suit, are able to compensate quite well to acceleration levels less than or equal to +7 GZ. Exposure to +9 GZ often resulted in unstable cardiovascular states involving relative bradycardia, often progressing to asystole, declining aortic blood pressure, and markedly diminished cardiac outputs approaching zero. It was found that, if aortic pressure and heart rate attain a relatively steady state during acceleration, and if heart level mean aortic pressure is greater than or equal to 100 Torr, the application of the microsphere technique during +GZ acceleration is theoretically valid. This hypothesis was tested using the microsphere technique (9.0 +/- 0.8 microns diam) in conscious miniature swine during exposure to +GZ acceleration. It is concluded that within the defined limits the radiolabeled microsphere technique is as accurate for use during acceleration studies as it is for use in routine laboratory studies.     

Treatment of hypertonicity in muscles of lip retraction

An EMG biofeedback program was developed for a 56-year-old Parkinsonism patient who exhibited pathological lip hypertonia and retraction. The program was designed to achieve the following goals: (1) to demonstrate a reduction in postural lip hypertonicity and (2) to demonstrate a reduction in lip hypertonicity during a series of increasingly complex speech activities. To achieve the first goal, contrastive tasks of full contraction and relaxation were utilized. Each posture was sustained while voltage measurements were made at specifici intervals. Procedures to modify lip retraction during speech included five tasks in which the patient was to monitor the audio feedback signal. The tasks involved: prolongation of a neutral vowel, consonant-vowel combinations, monosyllabic words, sentences, and a paragraph-reading task. Data collected over six biofeedback sessions are presented. Trend analyses showed consistent muscular reduction within each task. The following explanations for the decrease in the patient's hypertonicity were discussed: (1) reduction of anisometric contraction, (2) reduction of isometric contraction, (3) relearning of agonistic-antagonistic muscle balance.This research was supported in part by the University of Tennessee Neuropathology Services Program.     

Carbohydrate preferences of mammalian cells.

Carbohydrate preferences of mammalian cells can be utilized to biochemically distinguish between different cell lines. Ninety-three carbohydrates were examined of which (a) 15 supported cell proliferation and (b) 42 were toxic or growth inhibitory. The present investigation has employed an enzymatic system to eliminate trace glucose levels from reagents and glucose generated by serum enzymes.     

Functional heterogeneity among the T-derived lymphocytes of the mouse. IV. Nature of spontaneously induced suppressor cells.

When spleen cells are cultured for 4 days in the presence of fetal bovine serum, T-cells are generated which nonspecifically suppress the humoral responses of non-precultured cells to a variety of antigens. These T cells can be generated from both short-lived, sessile T1 cells and long-lived, recirculating T2 cells, and thus appear similar to suppressor T cells activated by concanavalin A.     

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.