Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir.

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv(r) clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv(r) mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.

Quality of life and psychological adaptation after surgical treatment for localized renal cell carcinoma: impact of the amount of remaining renal tissue.

OBJECTIVES: To analyze the quality of life and psychological adjustment after surgical therapy for localized renal cell carcinoma. METHODS: Postal questionnaires including measures of quality of life (SF-36) and the impact of the stress of cancer (Impact of Events Scale) were completed by 97 patients who had undergone radical or partial nephrectomy for localized renal cell carcinoma. Data were analyzed for the group as a whole and comparing the partial nephrectomy and radical nephrectomy groups. The variables examined included the impact of the type of partial nephrectomy (elective versus mandatory) and the amount of self-reported renal tissue remaining. RESULTS: The quality of life for the group as a whole was good, with no significant differences between the sample and U.S. norms for an age and sex-matched community sample on both the mental and physical health composite scores. Having undergone a partial versus a radical nephrectomy did not influence the patients' overall quality of life. Multiple linear regression modeling demonstrated that having more remaining renal parenchyma was an independent predictor of better self-reported physical health on the SF-36 (P <0.001). The entire sample had low mean scores on both avoidance and intrusion on the Impact of Events Scale, suggesting a lack of daily anxiety about cancer. Multiple linear regression modeling showed that patients who reported having more remaining renal parenchyma had lower intrusion and avoidance scores (P = 0.002 and 0.01, respectively). Multiple logistic regression modeling also demonstrated that the patients' perception of their remaining renal parenchyma was associated with less concern about cancer recurrence (P = 0.018) and less impact of cancer on patients' overall health (P <0.001). CONCLUSIONS: Most survivors of localized kidney cancer have normal physical and mental health regardless of the type of nephrectomy performed. The quality of life is better for patients with more renal parenchyma remaining after surgery for localized renal cell carcinoma.

Phase II trial of neoadjuvant estramustine and etoposide plus radical prostatectomy for locally advanced prostate cancer.

OBJECTIVES: To report the results of a Phase II trial of neoadjuvant estramustine and etoposide before radical prostatectomy in patients with locally advanced disease. METHODS: Treatment consisted of three cycles of estramustine (10 mg/kg/day) and etoposide (50 mg/m(2)/day) orally on days 1 through 21, repeated every 28 days, followed by radical prostatectomy. The eligibility criteria included locally advanced prostate cancer (clinical Stage T2b/c or T3, prostate-specific antigen [PSA] level of 15 ng/mL or greater, or Gleason score of 8 or higher) without evidence of metastatic disease. The median PSA level was 14 ng/mL (range 5.3 to 50), the median Gleason score was 7 (range 6 to 9), and 44% had Stage T2b/c or T3 disease. The primary endpoint was feasibility of neoadjuvant therapy and radical prostatectomy, including drug and surgery-related toxicities. Secondary endpoints included the pre-prostatectomy PSA level, local response, pathologic outcomes, and time to PSA failure. RESULTS: Eighteen patients were entered and completed all three cycles of therapy, and 16 (89%) underwent radical prostatectomy. A local response occurred in 15 (94%) of 16 patients with palpable tumors, and the serum PSA reached undetectable levels after therapy and before radical prostatectomy in 9 patients (50%). Five patients (28%) experienced grade 3 toxicity (two with deep venous thrombosis, two with neutropenia, and one with diarrhea) and one (6%) experienced grade 4 toxicity (pulmonary embolus) before surgery. The median operative time was 125 minutes, the mean blood loss was 665 mL, and the mean length of stay was 2.5 nights. Five minor surgical complications occurred in 4 patients. The pathologic analysis demonstrated residual carcinoma with squamous metaplasia and androgen deprivation effect in all patients. Five patients (31%) had organ-confined disease and 9 patients (56%) had specimen-confined disease. All patients achieved an undetectable PSA level postoperatively and at a median follow-up of 14 months (range 5 to 20) and without additional therapy, all 14 patients with negative lymph nodes were disease free. CONCLUSIONS: This trial confirms the feasibility of radical prostatectomy with acceptable surgical morbidity after neoadjuvant therapy with estramustine and etoposide in patients with locally advanced prostate cancer. However, this regimen is associated with estramustine-induced thromboembolic toxicity. The results of the pathologic analysis suggest a higher than expected rate of organ-confined and specimen-confined disease, but little histologic evidence of antitumor effect beyond that associated with androgen deprivation. Additional study of this paradigm with other drug regimens is warranted.

Surgery for invasive bladder tumors: technique and outcome.

The operative management of invasive transitional cell carcinoma has advanced significantly in the past year, particularly with respect to continent urinary diversion. The long term safety and efficacy of this form of urinary reconstruction is being established in terms of both operative and metabolic complications. The availability of continent diversion can decrease the interval to cystectomy and therefore may impact positively on survival. It has also been shown that continent diversion can safely be offered to patients at high risk for local recurrence. The importance of urethral sensory threshold on postoperative continence is being established. These findings and others continue to enhance the survival and quality of life of patients undergoing cystectomy for invasive bladder cancer.

Renal cell carcinoma-derived gangliosides suppress nuclear factor-kappaB activation in T cells.

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.

Mechanisms of apoptosis in T cells from patients with renal cell carcinoma.

Tumors may escape immune recognition and destruction through the induction of apoptosis in activated T lymphocytes. Results from several laboratories suggest that FasL (L/CD95L) expression in tumors may be responsible for this process. In this study of patients with renal cell carcinoma (RCC), we provide evidence for two mechanisms of T-cell apoptosis. One mechanism involves the induction of apoptosis via FasL expression in tumor cells. This is supported by several observations, including the fact that tumor cells in situ as well as cultured cell lines expressed FasL mRNA and protein by a variety of techniques. The FasL in RCC is functional because in coculture experiments, FasL+ tumors induced apoptosis in Fas-sensitive Jurkat T cells and in activated peripheral blood T cells but not in resting peripheral blood T cells. Most importantly, antibody to FasL partially blocked apoptosis of the activated T cells. Moreover, Fas was expressed by T cells derived from the peripheral blood (53% median) and tumor (44.3% median) of RCC patients. Finally, in situ staining for DNA breaks demonstrated apoptosis in a subset of T cells infiltrating renal tumors. These studies also identified a second mechanism of apoptosis in RCC patient peripheral T cells. Whereas these cells did not display DNA breaks when freshly isolated or after culture for 24 h in medium, peripheral blood T cells from RCC patients underwent activation-induced cell death after stimulation with either phorbol 12-myristate 13-acetate/ionomycin or anti-CD3/CD28 antibodies. Apoptosis mediated by exposure to FasL in tumor cells or through T-cell activation may contribute to the failure of RCC patients to develop an effective T-cell-mediated antitumor response.

13-year experience with percutaneous management of upper tract transitional cell carcinoma.

PURPOSE: We determined the immediate and long-term results of percutaneous management of upper trace transitional cell carcinoma in regard to rates of tumor recurrence and preservation of renal function. MATERIALS AND METHODS: Since July 1985, 12 men and 5 women 50 to 86 years old (mean age 72.2) years old underwent percutaneous management of upper tract transitional cell carcinoma. Of the patients 12 (71%) had a solitary kidney and 1 was treated bilaterally. In 16 of the 18 treated renal units (89%) definitive percutaneous resection of the tumor was followed by 6 weekly percutaneous installations of bacillus Calmette-Guerin. RESULTS: Complete resection was accomplished in 17 of the 18 renal units. Of the 18 renal units 15 (83.3%) had documented stage pTa lesions and 14 (77.8%) had grade 1/3 or 2/3 disease. Followup for all patients ranged from 1.7 to 75.5 months (mean 20.5). At the latest followup 11 patients (64.7%) are alive with no evidence of disease, and 6 (35.3%) died, 3 of whom (17.6%) had metastatic transitional cell carcinoma. Of the 13 patients undergoing treatment to solitary kidneys or bilaterally followup ranged from 1.7 to 75.5 months (mean 23.6). Serum creatinine ranged from 1.1 to 3.5 mg./dl. (mean 1.6) before percutaneous tumor resection and from 1.1 to 2.2 mg./dl. (mean 1.6) at the latest followup. Only 1 of these 13 patients (7.7%) with a solitary kidney has required dialysis. Ipsilateral local recurrence developed in 6 of the 18 renal units (33%), and in 4 of these 6 patients (67%) the tumor was grade 2/3 or 3/3 at initial resection. These recurrences were treated endoscopically in 4 patients, 3 of whom are currently without evidence of disease, and with nephroureterectomy in 2. Of the 17 patients only 1 (5.9%) with high grade (3/3), invasive (pT2) primary tumor at initial resection died of locally persistent or recurrent disease. CONCLUSIONS: Percutaneous management of upper tract transitional cell carcinoma is technically feasible and applicable in a significant number of patients in whom nephron sparing management is otherwise warranted. In carefully selected patients the results are at least comparable to other forms of "conservative" management in terms of tumor control and preservation of renal function.

A review of scrotal violation in testicular cancer: is adjuvant local therapy necessary?

High inguinal orchiectomy is the standard initial treatment for suspected testicular carcinoma. Nonstandard surgical approaches (scrotal violations), including scrotal orchiectomy, open testicular biopsy and fine needle aspiration, have historically been condemned as significantly compromising patient prognosis. Patients with scrotal violation are often subjected to potentially morbid or disfiguring local therapies. In addition, patients with scrotal violations are usually disqualified from surveillance protocols. A review was conducted of all published series of testicular cancer patients in whom scrotal violation occurred. A meta-analysis was then performed to choose a subset for critical analysis on the effect of scrotal violation on patient prognosis. Of 1,182 cases included in the final analysis scrotal violation occurred in 206. The rates for local recurrence, distant recurrence and survival were analyzed separately for all patients, patients with stage I disease and patients with pure seminoma or nonseminomatous germ cell tumor. Additionally, the effect of local treatment for scrotal violation on prognosis was examined. Although statistically significant differences were found in the local recurrence rate among the scrotal violation and inguinal group studies, the overall local recurrence rates were small (2.9% versus 0.4%, respectively). There were no statistical differences in distant recurrence or survival rates in all groups analyzed. Patients with scrotal violation who did not receive any local therapy fared as well as those who did receive local therapy. Although the standard treatment of primary testicular cancer remains high inguinal orchiectomy, these data suggest that scrotal violation does not impart a significantly worse overall prognosis. These data would also indicate that patients with stage I disease and scrotal violation should not necessarily be disqualified from surveillance protocols or subjected to adjuvant local therapy.

Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage.

The regulation of proteoglycan synthesis in a fibrocartilaginous tissue by mechanical loading was assessed in vitro. Discs of bovine tendon fibrocartilage were loaded daily with unconfined, cyclic, uniaxial compression (5 s/min, 20 min/day) and the synthesis of large and small proteoglycans was measured by incorporation of [35S]sulfate. All discs synthesized predominantly large proteoglycan when first placed in culture. After 2 weeks in culture nonloaded discs synthesized predominantly small proteoglycans whereas loaded discs continued to produce predominantly large proteoglycan. The turnover of 35S-labeled proteoglycan was not significantly altered by the compression regime. Increased synthesis of large proteoglycans was induced by a 4-day compression regime following 21 days of culture without compression. Inclusion of cytochalasin B during compression mimicked this induction. Autoradiography demonstrated that cell proliferation was minimal and confined to the disc edges whereas 35S-labeled proteoglycan synthesis occurred throughout the discs. These experiments demonstrate that mechanical compression can regulate synthesis of distinct proteoglycan types in fibrocartilage.

Small proteoglycan synthesis by skin fibroblasts cultured from elderly donors and patients with defined defects in types I and III collagen metabolism.

The relative synthesis of two different types of small proteoglycans with potentially distinct roles in tissue function (PGI and PGII) was investigated in human skin fibroblast cultures initiated from donors of increasing age (fetal to 92 y) and from patients with defined defects in type I and type III collagen metabolism. Because these two small proteoglycans are not distinguished by the usual methods of ion-exchange and sieve chromatography, we have separated them using gel electrophoresis and confirmed this by specific immunoprecipitation. Small proteoglycans of the PGII type were the predominant species found in the medium of all cultures from normal donors, regardless of age. Most of the mutant cell lines showed a profile of small proteoglycan synthesis like that of the normal cells (i.e., predominantly PGII) although an increased ratio of PGI/PGII was seen for two cell strains from patients with Ehlers-Danlos syndrome type IV characterized by intracellular accumulation of type III procollagen. We conclude that mutations affecting collagen primary structure and secretion appear to have little effect on the cells' synthesis and secretion of small proteoglycans. These findings fail to support an hypothesis suggesting that the metabolism of normal cellular synthetic products (proteoglycans) is altered by abnormal cellular processing of a defective product (collagen).

Increasing gene expression in yeast by fusion to ubiquitin.

Heterologous gene expression in yeast can be increased up to several hundred-fold by expressing a foreign gene as a fusion to the ubiquitin gene. An endogenous yeast endoprotease (Ub-Xase) removes the ubiquitin from the fusion product to produce the authentic protein. The utility of this technique has been demonstrated by expression of three different proteins in yeast as both unfused and ubiquitin-fused forms: 1) the alpha subunit of the mammalian stimulating G-protein of the adenylate cyclase complex (Gs alpha); 2) a soluble fragment of the T cell receptor protein (sCD4); and 3) the protease domain of human urokinase (UKP). The sequence specificity of the Ub-Xase was demonstrated by mutagenesis of the carboxyl-terminal glycine of ubiquitin to an alanine, which inhibited ubiquitin removal in vivo. Processing of the ubiquitin-Gs alpha fusion protein (ub-Gs alpha) in vivo resulted in Gs alpha which could be reconstituted in mammalian membrane preparations and had the same specific activity as the authentic Gs alpha expressed in yeast. The yeast Ub-Xase has also been shown to work in vitro by the processing of a ub-sCD4 fusion protein synthesized in Escherichia coli. This technology should greatly enhance the utility of yeast for heterologous protein production.

The modulating effects of lipids on purified rat liver Golgi galactosyltransferase.

Galactosyltransferase was purified from rat liver Golgi membranes. The Triton X-100, used to solubilize the enzyme was removed immediately prior to the lipid interaction studies. In lipid vesicles, prepared from a variety of phosphatidylcholines (PCs), including egg PC, DOPC, DMPC, DPPC and DSPC, the ability of the lipids to stimulate the enzyme decreased in the order egg PC greater than DOPC greater than DMPC greater than DPPC greater than DSPC, i.e. the lower the transition temperature (Tc) the greater the stimulation of the enzyme. A second, neutral lipid, phosphatidylethanolamine was used to permit a comparison of the effect of a different head group of the same net charge at neutral pH. The PEs included, egg PE, soy PE, Pl-PE, PE(PC) and DPPE in order of increasing Tc. The effect of the PEs was opposite to that of the PCs, i.e. the higher the Tc, the greater the stimulation of the enzyme. In fact egg PE and soy PE which have the lowest Tc values were inhibitory. Thus the modulation of the Golgi membrane galactosyltransferase by these lipids was different from that reported earlier for the bovine milk galactosyltransferase. The effects of two acidic lipids, egg phosphatidic acid (PA) and egg phosphatidylglycerol (PG) were studied also. Both totally inhibited the enzyme even at low concentrations of lipid, however, the PA was more effective than PG. In mixtures of neutral lipid (PC) and acidic lipid (PA or PG), the effect of the acidic lipid dominated. Even in the presence of excess PC, total inhibition of the enzyme was observed. It was concluded that the enzyme bound the acidic lipid preferentially to itself. The choice of the lipids allowed us to make several direct comparisons concerning the effect of the nature of the lipid head group on the activity of the enzyme. For example PE(PC), egg PA and egg PG would have fatty acid chains identical to egg PC since these three lipids are all prepared by modification of egg PC. As well, DPPE differs from DPPC only by nature of the head group. These comparisons indicated that not only the net charge but also chemical nature of the head group were important in the lipid modulation of Golgi galactosyltransferase.

Regulation of the yeast metallothionein gene.

To study regulation of the yeast CUP1 gene, we have employed plasmids containing the CUP1 regulatory sequences fused to the Escherichia coli galK gene. A comparison of galK expression from low- and high-copy-number CUP1/galK fusion plasmids demonstrated that both basal and induced levels of galactokinase (GalK) increase proportionately with plasmid copy number. Host strains with an amplified, single or deleted CUP1 locus were compared to look for effects of chromosomal CUP1 gene dosage on expression from the episomal CUP1 promoter. Basal GalK levels are similar in CUP1R and cupls hosts, but can be induced to higher levels in the cup1s than the CUP1R host. In contrast, in a strain deleted for the chromosomal copy of CUP1, synthesis of GalK is constitutive but can be induced to yet higher levels by copper. A hybrid vector, placing the CUP1 coding sequence under the control of a constitutive promoter, was constructed. Introduction of this hybrid CUP1 gene into the deletion host containing the CUP1/galK plasmid restores regulation. Thus, metallothionein, in trans, can effect repression of the CUP1 promoter. The possible roles of metallothionein and free copper in CUP1 regulation are discussed.

Therapeutic touch: is there a scientific basis for the practice?

The research-related literature on the topic of therapeutic touch is critically reviewed. The purpose of the review is to explore the current scientific basis for the teaching and practice of therapeutic touch as a treatment modality. An examination of published research literature indicates that empirical support for the practice of therapeutic touch is, at best, weak. The results of well-designed, double-blind studies have been transient, of no significance, or are in need of independent replication. Current practice of therapeutic touch is empirically little more than practice of placebo. Considerations for further nursing research are also presented.

Mössbauer spectroscopic studies of hemerythrin from Phascolosoma lurco (syn. Phascolosoma arcuatum).

Hemerythrin from coelomic cells of Phascolosoma lurco (syn. P. arcuatum) was isolated by gel filtration as two components, hemerythrin-I (25%) and hemerythrin-II (75%). The Mössbauer spectrum of oxyhemerythrin-II consisted of two pairs of lines of the same isomer shift (0.5 mm s(-1) corresponding to Fe(III) but different quadrupole splitting (1.01 and 2.02 mm s(-1). Application of a 2.5-T magnetic field at 4.2 K caused no significant spectral broadening. The 2FE.O2 binding site thus contains two nonequivalent high-spin Fe(III) ions that are antiferromagnetically coupled. The Mössbauer spectra of the minor component, hemerythrin-I, indicated an identical binding site. On deoxygenation, the spectrum was dominated by a simple quadrupole split doublet corresponding to Fe(II), indicating that the binding site in this derivative contains two identical Fe(II) ions that interact only weakly, if at all. The Mössbauer spectra of azidohemerythrin-II indicated that this derivative also contains a pair of antiferromagnetically coupled Fe(III) ions with the same isomer shift (0.5 mm s(-1)) but quadrupole splittings (1.40 and 1.96 mm s(-1)) that are not identical with those in oxyhemerythrin.