Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels.

Signal transduction in vertebrate rod and cone photoreceptor cells involves ion channels that are directly gated by the internal messenger cGMP. Rods and each type of cones express genetically related yet different forms of photopigments. Enzymes that control the light-stimulated hydrolysis of cGMP in rods and cones are also the product of distinct genes. Two different cDNA clones encoding cGMP-gated channels have been characterized from the chicken retina. Expression of cDNAs in Xenopus oocytes gives rise to cGMP-stimulated channel activity. Antibodies against a synthetic peptide specific for the C-terminal amino acid sequence derived from one clone stain outer segments of cone but not rod photoreceptors. Therefore chicken rod and cone cells each express different forms of cGMP-gated channels that are genetically related to each other. Expression in COS-1 cells produces the complete form of both channel polypeptides, whereas Western blot analysis indicates that channels in outer segment membranes are present in a processed form that is significantly shorter than the full-length polypeptide.

A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated channel from rod photoreceptor.

The cyclic nucleotide-gated channel from rod photoreceptors is composed of two distinct subunits (alpha and beta). The properties of the alpha subunit, which can form functional channels by itself, are modified by coexpression with a homologous polypeptide, designated the beta subunit. However, the alpha subunit from rod photoreceptor membranes copurifies with a 240 kDa protein that is significantly larger than this putative beta subunit. We now demonstrate by peptide sequencing and by cloning and functional expression of cDNA that the 240 kDa protein represents the complete beta subunit with an unusual bipartite structure. The N-terminal part is essentially identical to a glutamic acid-rich protein (GARP), whereas the C-terminal part is highly homologous to the previously cloned human "beta subunit." Expression of the complete beta subunit in HEK 293 cells results in a polypeptide with the same apparent molecular weight as the 240 kDa protein of the native rod channel. Coexpression of the alpha subunit with the full-length beta subunit yields hetero-oligomeric channels with properties characteristic of the native channel.

Controlled trial of megestrol acetate for the treatment of cancer anorexia and cachexia.

Preliminary information has suggested that megestrol acetate leads to appetite stimulation and nonfluid weight gain in patients with breast cancer, other cancers, and AIDS. Pursuant to this, we developed a randomized, double-blind, placebo-controlled trial of megestrol acetate in patients with cancer-associated anorexia and cachexia. We randomly assigned 133 eligible patients to receive 800 mg of megestrol acetate per day or a placebo. Patients assigned to megestrol acetate more frequently reported improved appetite (P = .003) and food intake (P = .009) when compared with patients receiving the placebo. A weight gain of 15 lb or more over baseline was seen in 11 of 67 (16%) patients receiving megestrol acetate compared with one of 66 (2%) given the placebo (P = .003). Patients receiving megestrol acetate reported significantly less nausea (13% vs. 38%; P = .001) and emesis (8% vs. 25%, P = .009). No clinically or statistically significant toxic reactions were ascribed to megestrol acetate, with the exception of mild edema. This study convincingly demonstrated that megestrol acetate can stimulate appetite and food intake in patients with anorexia and cachexia associated with cancer, leading to significant weight gain in a proportion of such patients.

Body-composition changes in patients who gain weight while receiving megestrol acetate.

PURPOSE: Randomized placebo-controlled clinical trials have now established that megestrol acetate causes appetite stimulation and weight gain in patients with anorexia and/or cachexia. There is a paucity of available data to delineate the substance of this increased weight. PATIENTS AND METHODS: Using dual-energy x-ray absorptiometry and tritiated body water methodologies, we performed body-composition measurements in 12 patients with advanced cancer before the institution of oral megestrol acetate (800 mg/d) and at subsequent 2-month intervals. RESULTS: Seven of the 12 patients gained weight (2.1 to 16.5 kg) and had repeat body-composition measurements performed at the time of maximum weight gain. The vast majority of the gained weight was clearly from an increase in adipose tissue, while there was a suggestion that an increase in body fluid was responsible for a minority of the weight gain. CONCLUSION: Megestrol acetate-induced weight gain is primarily the result of an increase in body mass.

Inhibition of fluorouracil-induced stomatitis by oral cryotherapy.

Mucositis is a significant dose-limiting toxicity associated with fluorouracil (5FU), particularly when it is combined with leucovorin. We hypothesized that oral cryotherapy would cause local vasoconstriction and would temporarily decrease blood flow to the oral mucous membranes. If cryotherapy were used during the time of peak serum 5FU levels, then the oral mucous membranes would have less exposure to 5FU and thus develop less mucositis. To test this hypothesis, 95 patients scheduled to receive their first cycle of 5FU plus leucovorin were randomized to have oral cryotherapy at the time of chemotherapy administration or to serve as a control group. Subsequent mucositis was significantly reduced in the group assigned to receive cryotherapy as judged by the attending physicians (P = .0002) and by the patients themselves (P = .0001). We now routinely recommend this cryotherapy procedure for our patients receiving daily bolus 5FU plus leucovorin.

Transdermal clonidine for ameliorating tamoxifen-induced hot flashes.

PURPOSE: To determine the efficacy of transdermal clonidine for alleviating tamoxifen-induced hot flashes in women with a history of breast cancer. PATIENTS AND METHODS: A randomized, double-blind, crossover design was used in this prospective study. Women with a history of breast cancer who were receiving tamoxifen and suffering from hot flashes were potentially eligible for this protocol study. RESULTS: Clonidine did reduce hot-flash frequency to a degree that was statistically impressive (P < .0001), but clinically moderate (20% reduction from baseline). It also decreased hot-flash severity (P = .02, 10% reduction from baseline). Clonidine was related to increased mouth dryness (P < .001), constipation (P < .02), itchiness under the patch (P < .01), and drowsiness (P < .05). CONCLUSION: Better means are needed to alleviate hot flashes among patients in whom estrogen therapy is contraindicated.

Trial of a topically administered local anesthetic (EMLA cream) for pain relief during central venous port accesses in children with cancer.

Procedure-related pain is a significant problem for many children receiving cytotoxic chemotherapy. In an effort to lessen this toxicity, we studied the efficacy and safety of administering topical local anesthesia using EMLA cream in 47 evaluable children with cancer undergoing implanted central venous port injections. Children (< 21 years old) scheduled to undergo repeated venous access procedures were selected for study. A placebo-controlled, randomized, double-blind, crossover study design was utilized. Statistically significant decreases in pain intensity scores (P < 0.002) were recorded by both children and investigators during the use of EMLA cream as compared with placebo. There was a good correlation between pain scores recorded by both patients and health care providers using both visual analog scales and categorized pain measurement tools. The topical application of EMLA cream 5% provides highly effective superficial anesthesia, and promises to be extremely useful for pain relief during percutaneous access procedures in cancer patients.

Epidemiologic features of pain in pediatric cancer patients: a co-operative community-based study. North Central Cancer Treatment Group and Mayo Clinic).

The prevalence, etiology, and management of pain in pediatric cancer patients seen at the Mayo Clinic and member institutions of the North Central Cancer Treatment Group were assessed. Participating centers, including both primary care and referral institutions, surveyed all patients seen during a 1-week period (Monday through Friday); procedure-related pain was excluded. Of the 160 children surveyed, 28 reported pain of which 57.8% was related to a side effect of anticancer treatment, 21.1% was unrelated to the malignancy, and 21.1% arose directly from the cancer. Pain intensity assessment was performed by both health-care professional and patient using a variety of measurement tools. Correlation between assessors was close except in young children. The predominance of treatment-related rather than cancer-related pain differs from results in series in adult cancer patients.

Biochemical constraints for survival under Martian conditions.

A wide variety of terrestrial organisms, the so-called "anhydrobiotes," has learned to survive in a state of extreme dehydration in dry environments. Strategies for survival include the accumulation of certain polyols and nonreducing saccharides, which help to prevent damage to membranes and proteins, but at low water partial pressure DNA is also progressively damaged by various lesions, including strand breaks and cross-linking to proteins. These lesions, if they are not too numerous, can be repaired before the first replication step after rehydration, but long-term exposure to dry conditions finally diminishes the chances of survival as these lesions accumulate. If an organism has no chance to repair the accumulated DNA damage during intermittent periods of active life, survival will not exceed a few decades. The restriction of survival by dryness-induced DNA lesions is corroborated by new data on conidia of Aspergillus and the free plasmid pBR 322. Our results will be discussed with respect to the chance of finding dormant life or biochemical fossils on the surface of Mars.

Survival in extreme dryness and DNA-single-strand breaks.

A wide variety of organisms (the so-called "anhydrobiotes') is able to survive long periods of time in a state of utmost dehydration and can thus survive in extremely dry environments including artificially imposed or space vacuum. Known strategies of survival include the accumulation of certain polyols, especially disaccharides, which help prevent damage to membranes and proteins. Here we report that DNA in vacuum-dried spores is damaged to a very substantial degree by processes leading to DNA strand breaks. Most of these lesions are obviously repaired during germination, but extensive damage to DNA and enzymes after long exposure times (months to years) finally diminish the chances of survival.

Extreme dryness and DNA-protein cross-links.

Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125 J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32P by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.

ERA-experiment "Space Biochemistry".

The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light. Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods. The decrease in viability of the microorganisms was as expected from simulation experiments in the laboratory. Accordingly, it could be correlated with the increase in DNA damages. The purple membranes, amino acids and urea were not measurably effected by the dehydrating condition of open space (in the dark). Plasmid DNA, however, suffered a significant amount of strand breaks under these conditions. The response of these biomolecules to high fluences of short wavelength solar UV-light is very complex. Only a brief survey can be given in this paper. The data on the relatively good survival of some of the microorganisms call for strict observance of COSPAR Planetary Protection Regulations during interplanetary space missions.

The symptom experience of mucositis, stomatitis, and xerostomia.

OBJECTIVE: To provide an overview of mucositis, stomatitis, and xerostomia, the symptom experience, risk factors, current therapies, nursing interventions, and implications for practice, research, and education. DATA SOURCES: Published articles pertaining to mucositis, stomatitis, and xerostomia. CONCLUSIONS: Complete and consistent assessment of the oral cavity are needed. Frequent and meticulous oral care remains an important factor in preventing the development of or reducing the severity of oral mucositis, stomatitis, and xerostomia. Additional research of these symptoms is needed. IMPLICATIONS FOR NURSING PRACTICE: Oncology nurses can perform an essential role in promoting oral hygiene and optimal status of the oral cavity during cancer and its treatment.

Cloning and chromosomal localization of a human class III myosin.

Two class III myosins have been identified to date: NINAC from Drosophila melanogaster and MyoIII(Lim) from Limulus polyphemus. Both have N-terminal kinase domains and are expressed exclusively in photoreceptors. Mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival. We report the cloning and chromosomal localization of a human class III myosin, MYO3A, from retina and a retinal pigment epithelial cell line. Human MYO3A (which we will refer to simply as MYO3A) possesses an N-terminal kinase domain and three consensus calmodulin-binding (IQ) motifs, two in the neck and one in the tail domain. We detected two MYO3A splice variants differing by 52 amino acids near the kinase/myosin junction. On Northern blots, MYO3A probes detected a 6. 5-kb transcript in human and monkey retina, in a cultured human RPE cell line (RPE-19), and at much lower levels in human pancreas. A somatic hybrid panel PCR screen localized MYO3A to human chromosome 10, and a radiation hybrid screen further localized it proximal to marker D10S197, which is located at 10p11.1 on the human cytogenetic map. Since mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival, the human homologue MYO3A may also play a role in photoreceptor function and/or maintenance.

Studies on the prevention of 5-fluorouracil-induced oral mucositis.

Oral mucositis is a major toxic effect related to 5-fluorouracil (5-FU) therapy. Clinical studies have attempted to identify an effective antidote for this untoward side effect. Early pilot studies suggested that an allopurinol mouthwash could lessen 5-FU-induced mucositis. However, a randomized, double-blinded, placebo-controlled crossover study did not suggest that an allopurinol mouthwash had any prophylactic value in this clinical situation. An ongoing, randomized clinical protocol is testing cryotherapy as a method of inhibiting 5-FU-induced stomatitis. No clinically appropriate prophylactic measure for preventing 5-FU-induced mucositis has been found to date.

Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane.

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.

Cloning of a novel C-terminal kinesin (KIFC3) that maps to human chromosome 16q13-q21 and thus is a candidate gene for Bardet-Biedl syndrome.

Kinesins are a large superfamily of microtubule motors that mediate specific motile processes. In a previous study, we identified 11 kinesin family members in the retina and retinal pigment epithelium (RPE) of the striped bass, Morone saxatilus. We have now identified, cloned, and sequenced the human homologue (KIFC3) of the most abundantly expressed retinal kinesin from that study, the C-terminal kinesin FKIF2. An antibody raised against an FKIF2 peptide cross-reacted with an approximately 80-kDa protein in human retina, RPE, kidney, and lung. Since microtubule-dependent processes are critical to the function and morphogenesis of the photoreceptors and RPE, the abundantly expressed KIFC3 was considered to be a potential candidate gene for causing human retinal degeneration. Chromosomal localization of the KIFC3 gene revealed that it maps to chromosome 16q13-q21, within the critical region for a Bardet-Biedl syndrome locus (BBS2). Bardet-Biedl syndrome is a genetically heterogeneous, autosomal recessive disorder characterized by retinal dystrophy, polydactyly, obesity, hypogonadism, renal abnormalities, and mental retardation. The chromosomal localization and expression pattern of KIFC3 suggest that it may be an excellent candidate for families linked to BBS2.

DNA-strand breaks limit survival in extreme dryness.

The inactivation of the anhydrobiotic organisms Bacillus subtilis (spores) and Deinococcus radiodurans during long-term exposure (up to several weeks) to extreme dryness (especially vacuum) is correlated with an increase in the number of DNA-strand breaks and other DNA lesions. Survival finally depends on the repair of DNA damages. Exposure of anhydrobiotic organisms to extreme dryness (e.g. on Mars or in space) for geological times will lead to so extended DNA lesions that recovery is extremely unlikely.

The cGMP-gated channel of the rod photoreceptor cell characterization and orientation of the amino terminus.

The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.