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Biomedical subjects

H Wolfe

Publications and source records attributed to H Wolfe.

At least 19 recordsLinked to original sources
PubMed1995

Mass spectrometric identification of amino acid transformations during oxidation of peptides and proteins: modifications of methionine and tyrosine.

Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS), tandem mass spectrometry with on-line liquid chromatography (LC/ESI-MS/MS) and high-resolution mass spectrometry with liquid secondary ionization (LSI-MS) were utilized to identify the modified amino acids in peptides and proteins formed during oxidation with performic acid. The procedure of protein oxidation was chosen to assist in protein unfolding by oxidizing the cystines to cysteic acids to allow for more complete proteolytic digestion and to create additional cleavage sites for endoproteinase Asp-N. Investigation of the Asp-N peptide map of oxidized superoxide dismutase (SOD) by LC/ESI-MS revealed that an expected proteolytic fragment of the protein was missing. In its place, two peptides with molecular weights 66 and 100 higher than that calculated for the missing peptide were observed. To identify the modified amino acids in the unexpected peptides, a model peptide with some amino acid similarities (tyrosine, arginine, methionine, lysine) to the missing peptide was chosen and was subjected to similar oxidation and enzymatic digestion steps, conditions, and reactions. After oxidation and digestion, the model peptide (TAP; sequence, Ac-MDKVLNRY) showed three major peaks in LC/MS. The peptides in the three peaks were identified as the unmodified peptide and two peptides whose molecular weights were 66 and 100 higher than that of TAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Expression of the ret proto-oncogene in phaeochromocytoma. An in situ hybridization and northern blot study.

The expression and distribution of ret proto-oncogene mRNA were investigated in five phaeochromocytomas of both familial and sporadic types by in situ hybridization (ISH) using digoxigenin-labelled cRNA probes and Northern blot (NB) analysis with random priming labelled cDNA probes. The probes corresponded to the tyrosine kinase domain of the gene. An excellent correlation was found between the ISH and NB results. By both techniques, the expression of the ret proto-oncogene was detected in three of the five cases, two of four familial tumours, and the only sporadic tumour that was studied. The results confirm that ret is frequently expressed in phaeochromocytomas and suggest that it might be an important event in their development and/or progression.

Adrenal Gland Neoplasms↗
PubMed1995

Tuberculosis knowledge among New York City injection drug users.

Structured interviews measuring tuberculosis knowledge were administered to 494 New York City injection drug users, 31% of whom reported a history of having a reactive tuberculin skin test. Medical records review of a subsample confirmed the validity of self-reported data. Most respondents understood the mechanisms of tuberculosis transmission. Three fourths of the subjects did not fully understand the distinction between a reactive skin test and active tuberculosis, but those who reported a history of skin test reactivity were twice as likely to understand this distinction. Forty percent of subjects did not understand the importance of medication adherence. Misunderstandings, based on a recent lack of tuberculosis education, may contribute to the fear and confusion that interfere with efforts to control tuberculosis.

Adolescent↗
PubMed1995

Tissue response to particulate polymethylmethacrylate in mice with various immune deficiencies.

We examined the tissue response to subcutaneous injections of particulate polymethylmethacrylate powder in fully immunocompetent C3Hf/Sed mice as well as three strains of mice with different levels of lymphocyte dysfunction. Five weeks after the injection, we found clearly demarcated granulomas. Histological and immunohistochemical studies showed that these granulomas were similar among all strains, with either paucity or absence of lymphoid cells. In situ hybridization with use of complementary RNA probes indicated that macrophages were synthesizing interleukin-1 beta messenger RNA (mRNA), a marker of macrophage activation, and a cytokine implicated in pathological bone resorption. We concluded that, in mice, there is a lymphocyte-independent pathway of macrophage activation in response to particulate polymethylmethacrylate. This suggests that the foreign-body response to particulate orthopaedic biomaterials is macrophage-initiated and maintained and that lymphocytes are not essential to this response, although they may modulate it.

Analysis of Variance↗
PubMed1994

Preparations for AIDS vaccine trials. Retention, behavior change, and HIV-seroconversion among injecting drug users (IDUs) and sexual partners of IDUs.

The likelihood that subjects in human immunodeficiency virus (HIV) vaccine efficacy trials will alter their behavioral risks for HIV infection over time must be considered in evaluating the feasibility of such trials and in estimating the necessary sample sizes to be enrolled. Potential subjects for future vaccine efficacy trials include injecting drug users (IDUs) and others who may be difficult to retain in studies and who may alter HIV-risk-related behaviors substantially over time. We have investigated behavior change, retention, and HIV seroconversion among 577 New York City resident IDUs and sexual partners of IDUs enlisted between July 1 and December 31, 1992. We attempted to see all subjects every 3 months for interviews, blood donation and HIV testing. We were able to retain 68% of subjects in the study through the third scheduled recall at 7.5-10.5 months after enlistment. HIV seroconversion through March 1, 1994, was 1.33/100 person-years at risk. There was a significant inverse relationship between HIV seroconversion and retention at the 9-month recall after adjusting for age, gender, and the amount of locator information provided by subjects at enlistment. Among subjects seen at each of the scheduled visits at 3, 6, and 9 months after enrollment, modest but statistically significant behavior changes that reduced risk were observed in self-reported drug injection frequency, heroin injection frequency, sexual contact with IDUs, and sharing of needles/syringes. The magnitude of these changes in risk, however, was small and may be transient. The behavior changes observed to date do not appear to be large enough to substantially alter calculations of sample sizes needed in future HIV vaccine efficacy trials.

AIDS Vaccines↗
PubMed1994

Intervening with high risk youth: a program model.

This paper describes a program model being implemented for older, high-risk adolescents who are beginning to exhibit significant substance use problems. The program provides an initial assessment and refers clients to appropriate services. Only a small percentage are being referred to inpatient treatment; most clients enter outpatient intervention groups, which are conducted by the program. These groups have three purposes: to allow staff to monitor and evaluate clients so that those who are in imminent danger can be placed in protective treatment settings; to help clients see themselves as candidates for recovery and to introduce them to treatment resources by accompanying them to AA and NA meetings; and to change behavior, a goal that seems more immediately achievable for youth with stable community ties who have more to lose if they get into further trouble. Besides describing the program model, this paper examines some of the difficulties of getting referrals from a variety of community agencies.

Adolescent↗
PubMed1992

Soft drink consumption and urinary stone recurrence: a randomized prevention trial.

The object of this study was to determine if a strong association between soft drink (soda) consumption and recurrence of urinary stone disease, found in an earlier case-control study of adult males, had a causal component. The study sample consisted of 1009 male subjects, who completed an episode of urinary stone disease, who were aged 18-75 at that time, and who reported consuming at least 160 ml per day of soft drinks. Half of the subjects were randomized to refrain from consuming soft drinks, while the remaining subjects served as controls. The intervention group had an observed 6.4% advantage in actuarial 3 yr freedom from recurrence (p = 0.023 one-sided) over the control group. One important secondary finding was that for those who reported at the time of the index stone that their most consumed drink was acidified by phosphoric acid but not citric acid, the experimental group had a 15% higher 3 yr recurrence-free rate than the controls, p = 0.002, while for those who reported at the time of the index stone that their most consumed drink was acidified by citric acid with or without phosphoric acid, the experimental group had a similar 3 yr recurrence-free rate to the controls, p = 0.55. This interaction was significant, p = 0.019.

Actuarial Analysis↗
PubMed1992

High-specificity in-situ hybridization. Methods and application.

We describe a technique of in-situ hybridization using oligonucleotide probes employing the expression of immunoglobulin VH genes as a model. Optimal conditions for hybridization with the 35S-labeled oligonucleotide probes were established with monoclonal B-cell lines that express VH genes of known nucleic acid sequence. The range of sensitivity and specificity achieved with this technique is documented. Under conditions of high stringency, this method can detect the expression of highly related VH hypervariable regions.

B-Lymphocytes↗
PubMed1992

The origins of hospital microcosting.

This paper begins with a definition of microcosting, including the specification of design criteria and a general model framework. The necessity for micro- as opposed to macro-costing is discussed. Examples of microcosting systems are provided. Relative value scale and case-mix alternatives to microcosting are presented.

Cost Allocation↗
PubMed1992

RURALSIM: the design and implementation of a rural EMS simulator.

Why were these applications of simulation technology unsuccessful? Parochialism, the volunteer nature of EMS planning, and limited regional commitment to resolve such complex problems at the local level all combined to present significant barriers to implementation. Political factors which posed the most significant barriers included: conservative attitudes concerning the funding and regulation of EMS activities by local governments; general opposition to governmental intervention in the private sector; strong resistance to mandatory standards for EMS; jurisdictional disputes between EMS-related agencies; lack of cooperation between the local governments; competition between prehospital-care providers and between hospitals; overemphasis on local jurisdictional boundaries in the planning and delivery of services; and the allocation of EMS resources, such as ambulances, according to political priorities, rather than more objective criteria. Based upon the results of the four field tests, the following observations are relevant: 1. RURALSIM is a very complex simulator. While every effort was made to assure generalizability, for any given situation, it required extensive modification and tailoring. The result was a model capable of handling a rather diverse set of situations, but one that could not be turned over to the general public for use. To implement RURALSIM required the participation of the University of Pittsburgh research team. The newer simulation languages now available alleviate this problem somewhat. 2. RURALSIM's complexity was needed to examine the different alternatives proposed by local planners. It was particularly needed in order to simulate each region's existing system. Such "base-line" simulations were required in order to achieve face validity and provide a basis for comparing alternatives. 3. With hindsight, a major weakness was the limited amount of face-to-face interaction between local planners and decision makers and the University of Pittsburgh staff. Only two trips to the region were budgeted. This proved to be insufficient and placed too much responsibility for model interpretation and analysis on the local contractors. 4. In none of the four test sites did the contractors and/or local health planners have the authority, influence and/or incentives necessary to develop regional EMS systems. In particular, none of the contractors were in the position to be decision makers, nor was there ever only one decision maker. This is not a criticism of the contractors, who did their best under difficult circumstances. Rather, it is a criticism of the state of eMS system development in the US in the early 1980s. There were few examples where regional systems developed successfully in the face of serious opposition from local interests.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Simulation↗
PubMed1991

Detection of K-ras mutations in pancreatic and hepatic neoplasms by non-isotopic mismatched polymerase chain reaction.

Mutations within codon 12 leading to activation of Kirsten-ras (K-ras) genes occur in a wide variety of human tumors, but have been reported most frequently in pancreatic carcinomas. We studied twenty-four paraffin-embedded pancreatic and hepatic tumors and two colon carcinoma cell lines with a rapid and simple approach that exploits allele-specific amplification of genomic DNA in a polymerase chain reaction (PCR). We extend the utility of this technique, which is dependent on an exact match at the 3' nucleotide between synthetic oligonucleotides and template DNA, to analyse paraffin-embedded tumor samples for the presence of point mutations at the first and second base of codon 12 of the K-ras gene. The PCR mismatch amplification technique demonstrated a 66% incidence of K-ras mutations at codon 12 in the group of pancreatic neoplasms as a whole. The percentage of mutations varied only slightly in the pancreatic cancer subcategories: 75% in ampullary, 66% in bile duct and 57% in the ductal adenocarcinomas. One islet cell carcinoma and normal tissues adjacent to the tumors revealed wild-type alleles only. One hepatoblastoma and one of six hepatocellular carcinomas also had codon 12 mutations. The PCR mismatch is a sensitive and rapid method that may be useful in screening neoplasms for K-ras point mutation and can be applied to archival material. This application allows a retrospective analyses of a wide range of pathological specimens to determine the role of K-ras mutations in human tumorigenesis.

Amino Acid Sequence↗
PubMed1991

Activated Val-12 ras p21 in cell culture fluids and mouse plasma.

Activation of ras oncogenes has been associated with a variety of cancers as well as their precursor lesions. Ras proteins activated by substitutions at amino acid positions 12, 13 or 61 have not been identified in normal tissues and therefore their detection may have clinical value. In this study our objective was to determine whether activated ras proteins could be released into the extracellular environment. To test this hypothesis, we used ras-transformed NIH3T3 cells that express an activated p21 containing valine (Val-12 p21) at position 12 instead of the normal glycine (Gly-12 p21) and a monoclonal antibody (mAb) designated DWP that is specific for the activated Val-12 ras proteins. Culture fluids collected from NIH3T3 cells transformed by the activated Val-12 p21 were shown, using mAb DWP in a sandwich ELISA format, to contain the activated Val-12 p21. In contrast, culture fluids from non-Val-12-containing cells were unreactive with mAb DWP. PSV-LM-EJ cells which overexpress the activated Val-12 p21 were injected subcutaneously (SQ) into nude mice to produce tumors. At the time of gross tumor appearance (14-21 days after tumor cell inoculation), plasma was collected from the PSV-LM-EJ tumor-bearing mice as well as from a series of control mice. Employing mAb DWP as a detection reagent in the sandwich ELISA format, we were able to detect the Val-12 p21 in the plasma of the PSV-LM-EJ tumor-bearing mice. Activated Val-12 p21 was not present in the plasma of non-tumor-bearing mice, or in the plasma of mice bearing SQ tumors composed of non-Val-12 p21 ras-transformed cells. This report is the first description of an activated ras protein (Val-12 p21) in the plasma of tumor-bearing mice and demonstrates that the results of the Val-12 p21-specific ELISA could be validated with Western blot format.

3T3 Cells↗
PubMed1990

Day-night variation in prepro vasoactive intestinal peptide/peptide histidine isoleucine mRNA within the rat suprachiasmatic nucleus.

Neurons within the suprachiasmatic nuclei of the hypothalamus (SCN) appear to function as a circadian clock that controls the timing of many physiological systems. The SCN contain several chemically distinct neuronal subpopulations, including a large group of interneurons within the ventrolateral SCN that exhibit co-localizable immunoreactivity for both vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The purpose of the present study was to determine whether VIP/PHI neurons within the rat SCN exhibit rhythmicity in the cellular levels of the messenger RNA encoding the precursor from which both VIP and PHI are derived. Using both quantitative in situ and solution hybridization prepro-VIP/PHI mRNA levels early in the dark phase were demonstrated to be significantly higher than those 5 h after the onset of the daily light period. Since no statistically reliable (P greater than 0.05) day-night variation was observed in the levels of prepro-VIP/PHI mRNA within cortex, these data suggest that the rhythmicity in prepro-VIP/PHI mRNA is an intrinsic property of VIP/PHI-containing SCN neurons, or rhythmically driven by local synaptic events within the SCN.

Animals↗
PubMed1990

Evaluation of potential early markers of chorioamnionitis associated with preterm premature ruptured membranes.

Optimal expectant management of preterm premature rupture of membranes (PROM) requires the early detection of chorioamnionitis. To date, however, no universally sensitive and specific marker for chorioamnionitis has been identified. Recently, the serial determination of plasma fibronectin, antithrombin, and prekallikrein has been reported to facilitate the early detection of sepsis in critically ill neonates and adult surgical patients. A cross-sectional study was undertaken to determine if plasma levels of these markers change significantly in patients with overt chorioamnionitis following expectant management of preterm PROM. Plasma levels of fibronectin and prekallikrein were not significantly different between the study (30 patients with overt chorioamnionitis following preterm PROM) and control (30 undelivered patients without antenatal complication matched for gestational age) groups. Antithrombin levels were significantly lower in the study group (p less than 0.05), but the magnitude of the difference (102% versus 94%) is not likely to be of clinical significance. We conclude that determination of plasma levels of fibronectin, prekallikrein, and antithrombin is not likely to aid in the early detection of chorioamnionitis in the setting of preterm (PROM).

Antithrombins↗
PubMed1990

Production and characterization of anti-RAS p21 monoclonal antibodies.

Monoclonal antibodies (MAb) Ras 10 and Ras 11 were raised to an activated human Harvey-ras p21 and shown to react with recombinant p21 as well as p21 derived from human and rodent cells. Characterization studies by ELISA, immunoprecipitation and Western blot procedures demonstrated that MAb Ras 10 (IgG2a) and Ras 11 (IgG2b) react with normal p21, activated p21, and p21 from each of the Harvey, Kirsten and N-ras families. Studies illustrated that MAb Ras 10 and Ras 11 can also be used in flow cytometry and immunohistochemistry to specifically detect cellular p21. ELISA, immunoprecipitation and Western blot studies comparing rat anti-p21 MAb Y13-259 with Ras 10 and Ras 11 demonstrated that Ras 10 and Ras 11 had a greater sensitivity for ras protein detection than Y13-259. Collectively, these studies illustrate that MAb Ras 10 and Ras 11 can be applied to a variety of assay formats to detect ras proteins and, therefore, may be valuable tools in detecting and measuring of ras protein expression in normal, neoplastic and pre-neoplastic cells.

Animals↗
PubMed1990

Comparison of measured and perceived time values for radiologists' work: impact on relative value scales.

The authors compared the actual time required by radiologists to perform and/or interpret common diagnostic radiologic examinations with the times the same radiologists perceived were necessary (as determined with a survey) to perform and/or interpret those same examinations. Average measured times ranged from 1.7 minutes for radiologic examinations of extremities to 113.2 minutes for interventional procedures. Average survey times ranged from 3.2 minutes for examinations of extremities to 84.4 minutes for cerebral angiography. The mean difference between measured and survey times for all examinations was 48.6%. Relative value scales were developed based on measured and survey times, with the upper gastrointestinal examination assigned the base unit of 100. The difference between the measured-time and survey-time relative value scales was 98% on average for the 16 examinations compared. The study suggests that there is a need for actual measurement of at least the time component of physician work if resource-based relative value scales are to be used as the basis for physician compensation.

Academic Medical Centers↗
PubMed1990

Melanotropin receptors demonstrated in situ in human melanoma.

Although some cultured human melanoma cell lines are responsive to melanotropins (melanocyte-stimulating hormones [MSH]), the prevalence and tissue distribution of MSH receptors in melanoma are unknown. We report here the use of an in situ binding technique to demonstrate specific MSH receptors in surgical specimens of human melanoma. The distribution and binding properties of specific MSH binding sites were determined by autoradiography and image analysis after incubation of frozen tumor tissue sections with a biologically active, radiolabeled analogue of alpha-MSH, [125I]iodo-Nle4, D-Phe7-alpha-MSH ([125I]NDP-MSH). In melanoma specimens from 11 patients, 3 showed high levels of specific binding, 5 showed low levels, and in 3 patients specific binding of [125I]NDP-MSH was not detectable. Specific MSH binding sites were present in melanoma cells, but not in adjacent connective or inflammatory tissues. Melanotropins, including alpha-MSH, NDP-MSH, and ACTH, inhibited [125I]NDP-MSH binding in a concentration-dependent manner, whereas unrelated peptides (somatostatin and substance P) did not. The apparent affinity of alpha-MSH for this binding site was in the nanomolar range (EC50 = 2 X 10(-9) M for inhibition of [125I]NDP-MSH binding in situ), similar to that recently described for the murine melanoma receptor. In one patient, analysis of multiple intratumor samples and tumors excised on three separate occasions revealed high levels of specific MSH binding in all samples. These results suggest that endogenous melanotropins may modulate the activities of human melanoma cells in vivo.

Autoradiography↗