Plasma bone-specific alkaline phosphatase as an indicator of osteoblastic activity

The total plasma alkaline phosphatase level has long been recognised as an indicator of osteoblastic activity, but lack of specificity makes it an insensitive index of the progress of disease and the response to treatment. Selective precipitation by wheatgerm lectin allows measurement of the plasma bone-specific alkaline phosphatase. We measured the plasma levels of this isoenzyme in 170 normal Chinese adolescents and adults, in 49 adults with fractures of a long bone, in 15 patients with osteosarcoma and in 38 patients with osteolytic metastases. The enzyme activity was also determined in 39 patients with liver disease. Of the patients with fractures, 94% had increased plasma activity during the healing process. The level was also increased in those with osteosarcoma but not in those with osteolytic bone metastases. There was no significant increase in activity in the patients with liver disease. We conclude that the plasma bone-specific alkaline phosphatase activity is a sensitive and reliable measure of osteoblastic activity.     

Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion

Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.     

Is urinary bladder pressure a sensitive indicator of intra-abdominal pressure?

Contribution of one case of right paratesticular rhabdomyosarcoma in a 3-year and 4-month old male patient. Following radical orchiectomy and clinical staging, grading is IRS Group I (fully resected localized disease). Subsequently, the patient received 7 polychemotherapy courses and was found to be asymptomatic 4 years after treatment.     

Viral dynamics of primary viremia and antiretroviral therapy in simian immunodeficiency virus infection

Mathematical modeling of viral replication dynamics, based on sequential measurements of levels of virion-associated RNA in plasma during antiretroviral treatment, has led to fundamental new insights into human immunodeficiency virus type 1 pathogenesis. We took advantage of the simian immunodeficiency virus (SIV)-infected macaque model to perform detailed measurements and mathematical modeling during primary infection and during treatment of established infection with the antiretroviral drug (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). The calculated clearance half-life for productively infected cells during resolution of the peak viremia of primary infection was on the order of 1 day, with slightly shorter clearance half-lives calculated during PMPA treatment. Viral reproduction rates upon discontinuation of PMPA treatment after 2 weeks were approximately twofold greater than those obtained just prior to initiation of treatment in the same animals, likely reflecting accumulation of susceptible target cells during treatment. The basic reproductive ratio (R0) for the spread of SIV infection in vivo, which represents the number of productively infected cells derived from each productively infected cell at the beginning of infection, was also estimated. This parameter quantifies the extent to which antiviral therapy or vaccination must limit the initial spread of virus to prevent establishment of chronic disseminated infection. The results thus provide an important guide for efforts to develop vaccines against SIV and, by extension, human immunodeficiency virus.     

Reproductive efficiency as a social indicator

An index of reproductive effiency (RE) is proposed as a social indicator that will meet the need to consider various forms of pregnancy wastage, to compare their relative costs, and to guide reproductive health policy accordingly. This article discusses conceptual and measurement aspects of RE. Conversion of wanted to unwanted pregnancies and the reverse, interpretation of abortion in relation to other pregnancy outcomes, defining the end point for the reproductive process and criteria for the events to be included as significant outcomes are conceptual issues. Measurement problems include: whether aggregation is justified, prospective and retrospective tracking of outcomes, record limitations, duplication of adversities in a single pregnancy, and selection of optimal rate for comparison. A measurement of RE for the entire United States based on the National Natality Survey of 1964-1966 is presented, showing 74.5 percent of pregnancies resulting in healthy liveborn infants. For those years, data on abortions could not be included. Within the group of reported pregnancy losses, the importance of congenital abnormalities and low-birth-weight babies is enhanced by application of economic weights based on associated medical care costs. Changing opportunities for birth timing, prenatal and infant care, and control of family size are social means of reducing adverse outcomes associated with teenage pregnancy and high-parity births, often found together with poverty. Successive increments in RE may be progressively more expensive to achieve, and cost effectiveness comparison will be necessary.     

Increased mitotic activity as a negative prognostic indicator in pleomorphic xanthoastrocytoma. Case report

Pleomorphic xanthoastrocytoma is a recently characterized neoplasm with a favorable prognosis despite aggressive histological features. The authors report a case of pleomorphic xanthoastrocytoma that recurred 4 years after complete gross resection. The original tumor exhibited histological features characteristic of this neoplasm, but up to 4 mitoses/10 high-power fields were present focally. The recurrent tumor contained small foci of classical pleomorphic xanthoastrocytoma, but consisted predominantly of glioblastoma multiforme. Transitional zones contained nests of glial fibrillary acidic protein (GFAP)-immunopositive cells surrounded by delicate collagenous and reticulin-rich septa. Electron microscopy of the transitional zone showed continuous basal lamina investing cells containing bundles of intermediate filaments. These were GFAP-positive by immunogold electron microscopy, confirming the astrocytic nature of pleomorphic xanthoastrocytoma. This example illustrates the capacity of this tumor to evolve into glioblastoma. The indolent clinical behavior of most pleomorphic xanthoastrocytomas is evident from a literature review, which confirms the prolonged survival of many patients after onset of symptoms. Completeness of excision, subjectively assessed at surgery, did not influence the risk of recurrence or survival up to 10 years after initial resection. Postoperative radiotherapy did not improve survival, but may reduce the probability of recurrence; more studies are needed to corroborate this finding. The data compiled herein support the designation of pleomorphic xanthoastrocytoma as a distinct astrocytic neoplasm with a favorable prognosis. An increased mitotic rate has not previously been correlated with a worse outcome, and should not be used to exclude this diagnosis. However, anaplastic transformation of pleomorphic xanthoastrocytoma confers a much worse prognosis, and this case suggests that increased mitotic activity may be a negative prognostic indicator since it may herald subsequent anaplastic transformation.     

delta-Aminolevulinic acid in plasma or whole blood as a sensitive indicator of lead effects, and its relation to the other heme-related parameters

To evaluate the subclinical effect of lead exposure, we determined delta-aminolevulinic acid (ALA) levels in plasma (ALA-P), blood (ALA-B), and urine (ALA-U) and the activity of delta-aminolevulinic acid dehydratase (ALAD) in lead workers. Almost all of the ALA molecules in blood were present in plasma and not in blood cells, irrespective of the blood lead concentration (Pb-B). ALA-P or ALA-B levels increased slowly at Pb-B levels below 40 micrograms/dl (slow phase) and rapidly at levels above 40 micrograms/dl (rapid phase). In both phases, ALA-P and ALA-B were well correlated with Pb-B and ALAD activity. The threshold value (no-effect level) of Pb-B for elevation of the ALA-P or ALA-B level was coincident with that for ALAD inhibition; the value was around 5 micrograms/dl. In the rapid phase, ALA-P increased continuously up to 100 micrograms/dl of Pb-B, while ALAD activity reached a plateau. Receiver operative characteristic (ROC) plot analyses indicated that ALA-P and ALAD activity [ALAD(u)] had a similar diagnostic value at Pb-B levels between 10 and 40 micrograms/dl, although ALAD(%), the remaining ALAD activity as a percentage of the whole activity restored by zinc and dithiothreitol, had the most powerful diagnostic efficiency at these Pb-B levels. By contrast, ALA-U and zinc protoporphyrin were less effective for the diagnosis of lead exposure than ALAD and ALA-P. These findings indicate that ALA-P is the best discriminators of lead exposure form baseline to high levels of exposure.     

Tryptophan requirement in young adult women as determined by indicator amino acid oxidation with L-[13C]phenylalanine

alpha-Difluoromethylornithine (DFMO) is a suicide inhibitor of ornithine decarboxylase and potent antiproliferative chemopreventive agent. We conducted a dose de-escalation Phase I trial of DFMO in patients with grade 3 cervical intraepithelial neoplasia to determine an optimal dose of DFMO using ornithine decarboxylase activity and polyamine modulation as surrogate biomarkers and to evaluate its toxicity. Thirty patients with biopsy-confirmed grade 3 cervical intraepithelial neoplasia were assigned sequentially to one of five DFMO doses (1.000, 0.500, 0.250, 0.125, or 0.060 g/m2) given daily for 31 days. One patient was excluded from analysis for protocol violation. Polyamine levels were assessed in cervical tissue, plasma, and RBCs. Tissue and blood samples were obtained before and after treatment with DFMO. All patients underwent loop excision of the cervix at the end of the study for complete histological evaluation and definitive treatment of the premalignant condition. No major clinical toxicity was observed at any DFMO dose. A reduction in tissue spermidine to spermine (SPD:SPM) ratio and an increase in plasma arginine levels were observed among patients receiving 1.000 g/m2/day (P < 0.05). A nonsignificant reduction in SPD:SPM ratio was also observed in the 0.500 g/m2/day dose group, and a nonsignificant increase in plasma arginine level was observed down to the 0.125 g/m2/day dose level. There was no evidence of modulation of other polyamines or precursors. Fifteen patients experienced a complete (5 patients) or partial (10 patients) histological response. In conclusion, DFMO was well tolerated and significantly modulated tissue SPD:SPM ratio and plasma arginine level at the dose of 1.000 g/m2/day. To clarify whether DFMO has activity at lower doses, these results will be tested in a three-armed double-blinded Phase II study using placebo and DFMO doses of 0.500 and 0.125 g/m2/day.     

99Tcm-MAG3: a sensitive indicator for evaluating perfusion and rejection of renal transplants

Radionuclide renography has a role in evaluating perfusion of transplanted kidneys. In the course of rejection, cortical perfusion decreases before urinary excretion changes. Based on the facts that 99Tcm-MAG3 has different pharmacokinetics and shows a higher kidney-to-background count ratio than 99Tcm-DTPA, we postulated that 99Tcm-MAG3 was a sensitive and reproducible agent to measure cortical perfusion of transplanted kidneys. To clarify the feasibility of using 99Tcm-MAG3 to measure the cortical perfusion index (CPI), sequential renography was performed using 99Tcm-DTPA and 99Tcm-MAG3 in 14 patients with stable renal transplants, who had changes in serum creatinine concentration of less than 50% between the two studies. The CPI was calculated with 99Tcm-DTPA and 99Tcm-MAG3 and these were then compared and correlated with concurrent serum creatinine concentration. The CPI with 99Tcm-MAG3 was 1.43 times that with 99Tcm-DTPA in patients with changes in serum creatinine concentration equal to or less than 20%, and regression analysis revealed that the difference in CPI was larger in patients with more severely decreased renal perfusion than in patients with normal or mildly decreased renal perfusion. This preliminary study has indicated that the CPI with 99Tcm-MAG3 is a sensitive index for detecting changes in renal function, and thus is a feasible indicator of cortical perfusion when evaluating the rejection of transplanted kidneys.     

Use of rectal cancer position as a prognostic indicator

The hypothesis of this study was that the position of rectal cancer within the circumference of the rectum influences mortality. Tumor position was prospectively documented in 181 patients with rectal carcinoma by two examiners. The results were analyzed for correlation to survival using the Lifetest model and for multivariate correlation using the Cox regression model. An anterior tumor location was present in 43 patients and was found to have a significantly higher survival rate than other positions. Two-thirds of anterior tumors were of pathologically favorable Dukes' stages. Fifty-five patients had posterior tumors with decreased survival rates, two-thirds of which were of unfavorable stages. Circumferential position in 61 patients was most predictive of poorer outcome, with a relative risk of death being increased by 4.6 times (P = 0.014) and a 5-year survival rate of 68.8 per cent; 85 per cent of these tumors were of pathologically unfavorable stages. The 5-year survival rate for the whole group, which included 181 patients with all histopathological stages except those with distant metastases at presentation, was 78.5 per cent. This ranking of survival rates was found to be consistent in each category with the postoperatively determined Dukes' stage, which carried a prognostic significance of P = 0.0001. We conclude that tumor position is a significant indicator of prognosis available before surgery for rectal cancer.     

Melatonin as a chemical indicator of environmental light-dark cycle

Melatonin (N-acetyl-5-methoxytryptamine) is an evolutionary highly conserved molecule that plays an important role in conveying the clock and calendar information to all living organisms, including man. Melatonin is synthesized in the rhythmic fashion, primarily by the pineal gland, and, to a lesser degree, by extrapineal tissues-namely the retina, the Harderian gland, and the gastrointestinal tract. The rhythm of the hormone production, with maximal levels occurring at night in darkness, is generated by an endogenous circadian clock(s) and is synchronized with the photoperiodic environment to which animals are exposed. This brief outline surveys data on the regulation of rhythmic melatonin biosynthesis by a circadian pacemaker and light (full spectrum white light and monochromatic lights with wavelengths both in the visible and invisible range). Additionally, possible applications of this chronobiotic compound in agriculture and in medicine in the treatment of circadian rhythm sleep disorders are discussed.     

Bufotenine reconsidered as a diagnostic indicator of psychiatric disorders

We have analyzed products of the serotonin-degradative pathway, in which both N-methylserotonin and bufotenine are formed in urine specimens of products with psychiatric disorders by three-dimensional HPLC with electrochemical detection. Bufotenine was detected in urine from all autistic patients with mental retardation and epilepsy (n = 18) and many autistic patients (32/47) with mental retardation. Bufotenine was detected in the urine of 15 of 18 patients with depression. Thirteen of 15 schizophrenic patients were also positive for bufotenine. N-methylserotonin was also detected in some cases of each disorder. Only two of 200 urine specimens from healthy controls were positive for bufotenine. Thus, the presence and levels of bufotenine might be useful and important markers of some psychiatric disorders.     

Autoimmunity, immunodeficiency and mucosal infections: chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of "molecular mimicry" (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.     

Green fluorescent protein as a noninvasive intracellular pH indicator

It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and approximately 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in <1 ms. At lower pH, the fluorescence response was slowed and not completely reversed. These findings suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.     

Measurement of carbonic anhydrase activity using a sensitive fluorometric assay

The dehydration reaction of bicarbonate was measured using the fluorescent pH indicator, 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), in combination with stopped-flow spectrofluorometry. The initial rate of bicarbonate dehydration was measured after mixing a pH 6.0 solution with a pH 8.0 solution containing bicarbonate. Addition of carbonic anhydrase to the pH 6.0 solution enabled the measurement of the initial rate of activity at physiological temperatures with resolution times of 2 ms. This assay was used to resolve differences in activity and sensitivity to sulfonamides by comparing mammalian carbonic anhydrase isoforms. The fluorescent technique used in this study is very sensitive, allowing the determination of initial rates with a protein concentration as little as 65 ng/ml. Pyranine can also be loaded into membrane vesicles to follow carbonic anhydrase activity within vesicles. The change in pH within vesicles is dependent on the concentration of externally added bicarbonate and the presence of carbonic anhydrase on either side of the membrane. Therefore, this assay can be used to measure carbon dioxide flux across membranes and to assess the contribution of carbonic anhydrase to this flux.     

Serum hepatocyte growth factor as a possible indicator of arteriosclerosis

OBJECTIVE: To investigate the possible involvement of hepatocyte growth factor in arteriosclerotic lesions, by studying the relationship between serum concentrations of hepatocyte growth factor and grades of retinal arteriosclerosis. METHODS: We measured the blood pressure, body mass index, serum concentrations of total cholesterol, high-density lipoprotein cholesterol, triglycerides, creatinine, uric acid, total protein, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, gamma-glutamyltranspeptidase, alkaline phosphatase, and hepatocyte growth factor, erythrocyte counts, hemoglobin concentration, and hematocrit levels of 112 adults. Serum concentrations of hepatocyte growth factor were measured by a specific enzyme-linked immunosorbent assay. For each subject, photographs of both optic fundi were taken, and the grade of arteriosclerotic changes in the retinal arteries was evaluated according to Scheie's classification. RESULTS: Individuals with more advanced grades of arteriosclerotic changes had higher serum hepatocyte growth factor values (grade 0, 0.056 +/- 0.004 ng/ml, n = 86; grade 1, 0.132 +/- 0.026 ng/ml, n = 17, P < 0.01, versus grade 0; grade 2-3, 0.271 +/- 0.023 ng/ml, n = 9, P < 0.01, versus grades 0 and 1). The serum hepatocyte growth factor concentrations were also correlated significantly to the serum uric acid concentrations (r = 0.230, P = 0.015) and erythrocyte counts (r = 0.299, P = 0.001), but not to the systolic and diastolic blood pressures, and other physical and humoral parameters. CONCLUSIONS: Serum hepatocyte growth factor levels are thought to indicate the presence or development of arteriosclerotic lesions and may be a useful biochemical parameter for estimating the development of systemic arteriosclerosis irrespective of blood pressure levels.