Characterization of the putative cholesterol transport protein metastatic lymph node 64 in the brain.

Intracellular management of cholesterol is a critical process in the brain. Deficits with cholesterol transport and storage are linked to neurodegenerative disorders such as Neimann-Pick disease type C and Alzheimer's disease. One protein putatively involved in cholesterol transport is metastatic lymph node 64 (MLN64). MLN64 localizes to late endosomes which are part of the cholesterol internalization pathway. However, a detailed pattern of MLN64 expression in the brain is unclear. Using immunocytochemical and immunoblot analyses, we demonstrated the presence of MLN64 in several tissue types and various regions within the brain. MLN64 immunostaining in the CNS was heterogeneous, indicating selective expression in discrete specific cell populations and regions. MLN64 immunoreactivity was detected in glia and neurons, which displayed intracellular labeling consistent with an endosomal localization. Although previous studies suggested that MLN64 may promote steroid production in the brain, MLN64 immunoreactivity did not colocalize with steroidogenic cells in the CNS. These results demonstrate that MLN64 is produced in the mouse and human CNS in a restricted pattern of expression, suggesting that MLN64 serves a cell-specific function in cholesterol transport.

Progression of transplanted SJL/J lymphomas attributed to a single aggressive H-2Ds-negative lymphoma.

Spontaneously arising, H-2Ds-positive SJL/J lymphomas have been reported to become irreversibly more aggressive and H-2Ds-negative upon successive transplantation in syngeneic mice. In an effort to determine whether this process was one of tumor progression, we sought to: (a) establish whether a clonal relationship exists between readily transplantable aggressive SJL/J lymphomas and their respective indolent predecessors; and (b) identify genetic events critical to the process of acquisition of increased malignancy. Examination of putatively distinct, aggressive, H-2Ds-negative lymphomas, including the long term transplantable line RCS5, revealed them to have the same heavy and light immunoglobulin chain gene rearrangement patterns, a characteristic karyotype marked by nine chromosomal abnormalities, and approximately ten newly acquired ecotropic murine leukemia proviruses at similar genomic sites. Independent, spontaneously arising H-2Ds-positive lymphomas, in early transplant, were found to be genetically distinct from the respective more malignant H-2Ds-negative tumors to which they gave rise during successive transplantation. The data are interpreted as indicating that the aggressive H-2Ds-negative tumors in this study originated from a common source, most likely the RCS5 tumor, rather than through progression of separate spontaneously arising SJL/J lymphomas. It cannot be concluded which of the multiple genetic abnormalities of the H-2Ds-negative tumors were critical to their highly malignant phenotype. However, chromosomal abnormalities and newly acquired murine leukemia proviruses are discussed as to the roles they might play in SJL/J lymphomas.

Loss of myb expression in an aggressive SJL/J B-cell lymphoma.

SJL mice spontaneously develop B-cell lymphomas that can be propagated by transplantation into syngeneic mice. These tumors usually have an indolent phenotype and require at least several weeks to produce morbidity following transplantation. However an aggressive lymphoma (RCS5) has been found that produces morbidity within days of transplantation. RCS5 cells fail to express the H-2Ds class I major histocompatibility complex antigen, whereas indolent tumors express H-2Ds. To identify genetic factors that may contribute to the tumorigenicity of B-cell lymphomas in SJL mice, tumor genomes were analyzed for mutations in cellular oncogenes. No rearrangements were detected by Southern hybridization analysis in tumors at the abl, myc, mbcl-2, Ha-ras, Ki-ras and raf loci. Indolent tumors were not rearranged at the myb oncogene, however alterations were detected in both myb alleles in RCS5. Northern hybridization analysis on RNA from in vivo-derived tumor preparations failed to detect any myb transcripts in RCS5. The loss of normal myb expression could directly contribute to the aggressive phenotype of RCS5. Alternatively, expression of the RCS5 myb allele may have contributed to early stages of tumor development. The possibilities that the observed myb mutations affect tumor aggressiveness and H-2Ds expression are discussed.

Selective loss of H-2Ds antigen on a murine B lymphoma due to a post-transcriptional block in expression.

The major histocompatibility (MHC) class I antigens are coordinately expressed in most cells. However, some tumors or virus-infected cells lack expression of one MHC class I antigen, while expression of the other MHC class I antigens is unaffected. We previously described the selective expression of MHC class I antigens on a B-cell lymphoma from SJL/J mice called RCS5. This tumor expresses H-2Ks, but has lost cell surface expression of H-2Ds. To understand the mechanism responsible for the selective loss of H-2Ds on the cell surface, we analysed H-2Ds mRNA and protein in the RCS5 tumor. Here we report that H-2Ds mRNA was expressed in RCS5, but H-2Ds protein was not detected in cell lysates. To determine whether the H-2Ds mRNA from RCS5 was able to direct the synthesis of H-2Ds protein, we performed cDNA cloning, in vitro translation and gene transfer experiments using a cell line related to RCS5 (cRCS-X). Our results indicated that the inhibition of H-2Ds expression in cRCS-X occurred after transcription of a non-defective H-2Ds mRNA. Furthermore, H-2Ds antigen expression was restored in cRCS-X using a retroviral vector to express the recombinant H-2Ds cDNA. These results indicate that the inhibition of H-2Ds expression could be overcome either by out competing an inhibitor that functions in trans or by removing cis-acting regulatory sequences from the endogenous H-2Ds mRNA.

Generation of retroviral vector for clinical studies using transient transfection.

Transient transfection of 293T cells was utilized to produce high-titer murine recombinant retroviral vectors for clinical studies. This system was initially optimized by gene transfer using different retroviral envelope proteins into activated human CD4+ T lymphocytes in vitro. Higher titer and infectivity were obtained than with stable murine producer lines; titers of 0.3-1 x 10(7) infectious units per milliliter for vectors encoding the green fluorescent protein (GFP) were achieved. Virions pseudotyped with envelope proteins from gibbon ape leukemia virus or amphotropic murine leukemia virus resulted in gene transfer of > or = 50% in CD4+ human T lymphocytes with this marker. Gene transfer of Rev M10 with this vector conferred resistance to HIV infection compared with negative controls in the absence of drug selection. Thus, the efficiency of transduction achieved under these conditions obviated the need to include selection to detect biologic effects in T cells. Finally, a protocol for the production of large-scale supernatants using transient transfection was optimized up to titers of 1.9 x 10(7) IU/ml. These packaging cells can be used to generate high-titer virus in sufficient quantities for clinical studies and will facilitate the rapid, cost-effective generation of improved retroviral, lentiviral, or other viral vectors for human gene therapy.

Implications of progesterone metabolism in MA-10 cells for accurate measurement of the rate of steroidogenesis.

In virtually all studies with MA-10 cells, progesterone RIAs have been used to measure steroid synthesis. To test whether progesterone is a stable end product, we investigated the metabolism of added tritiated progesterone and pregnenolone in MA-10 cells over a period of 3 h. Steroids were then extracted, separated by HPLC, and identified by GC/MS. We found that more than 70% of radiolabeled steroids were converted to at least five different metabolites. A major metabolite (40%) was 5 alpha-pregnan-3 alpha or 3 beta-ol-20one. Similar studies, using radiolabeled T, demonstrated conversion to dihydrotestosterone and two forms of 5 alpha-androstane-diols. These data indicate the presence of active 5 alpha-reductase and 3 alpha- and/or 3 beta-hydroxysteroid dehydrogenase activities in MA-10 cells. Because these results suggest that progesterone is an unstable end product, to gauge the level of active metabolism, we incubated cells in the presence of inhibitors of pregnenolone metabolism and assessed pregnenolone levels by RIA. We discovered that basal levels of steroidogenesis in MA-10 cells were considerably higher than previously estimated. Moreover, dibutyryl cAMP-stimulated steroid production was linear over more than 13 h, in contrast to previous findings that measured progesterone levels. Other consequences of inaccurate assessment of steroidogenic activity in MA-10 cells because of the application of the progesterone assay are discussed.

Steroid production after in vitro transcription, translation, and mitochondrial processing of protein products of complementary deoxyribonucleic acid for steroidogenic acute regulatory protein.

We have previously demonstrated that steroidogenic acute regulatory protein (StAR) is essential for the rate-limiting step in the acute regulation of steroidogenesis, which is the transport of cholesterol from the outer to the inner mitochondrial membrane. We have hypothesized that this transport occurs as the 37-kilodalton (kDa) precursor form of StAR is imported into the mitochondria and processed to its 30-kDa mature forms. Using an in vitro transcription and translation system in the presence of mitochondria isolated from unstimulated mouse MA-10 Leydig tumor cells, we now directly show that the 37-kDa form is indeed the cytosolic precursor of StAR and can be processed by mitochondria to all four 30-kDa mature forms. To determine the subcellular location of StAR in steroidogenic cells, ultrastructural immunocytochemistry was performed in adrenal zona fasciculata cells using the protein A-gold technique. We show that StAR is associated exclusively with the mitochondria. There, StAR is primarily localized in the intermembrane space and the intermembrane space side of the cristae membrane. StAR was shown to induce steroid production in isolated mitochondria. StAR protein was expressed in COS1 cells and the cell lysate, which was shown to contain abundant levels of StAR by Western blot analysis, was incubated with mitochondria isolated from unstimulated MA-10 cells. In these experiments, StAR increased steroid production by at least 4-fold over control mock-transfected lysate, and this increase was time and dose dependent. Furthermore, the increase in steroid production induced by StAR-containing lysate was not observed when COS1 lysate containing high levels of another mitochondrially imported protein, adrenodoxin, was used. We conclude from these results that in response to tropic hormone stimulation of steroidogenic cells, StAR is synthesized as a 37-kDa precursor, imported into the mitochondria, processed to its 30-kDa mature forms, and localized to the intermembrane space. During import and processing in vitro, StAR induces steroid production in isolated mitochondria in a specific manner.

Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary.

The steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein that is indispensable for the synthesis of steroid hormones in the steroidogenic cells of the adrenal cortex and the gonads. Recent studies have shown that StAR enhances the conversion of the substrate for all steroid hormones, cholesterol, into pregnenolone, probably by facilitating cholesterol entry into the inner compartment of the mitochondria where the steroidogenic cytochrome P450scc complex resides. To study the potential of StAR to affect ovarian steroidogenesis during follicular development, we examined the time-dependent expression of StAR protein and messenger RNA in PMSG/human CG (hCG)-treated immature rats. Western blot analyses and immunohistochemical and RT-PCR methodologies have revealed a biphasic expression of StAR in the ovaries responding to hormones. The first peak of StAR expression was generated by PMSG administration and lasted for 24 h. Furthermore, it was restricted to the entire network of the ovarian secondary interstitial tissue, as well as to a fewer scattered theca-interna cells. The second burst of StAR expression was observed in response to the LH surge, as simulated by hCG. This time, StAR was expressed in the entire theca-interna and interstitial tissue, as well as in those granulosa cells that were confined to periovulatory follicles. Immunoelectron microscopy studies revealed the over 90% of StAR antigenic sites are localized in the inner compartments of the mitochondrion, suggesting a rapid removal of StAR precursor from the mitochondrial surface, where it is believed to exert its activity. Altogether, our observations portray dynamic acute alterations of StAR expression during the process of follicular maturation in this animal model. Furthermore, if StAR indeed determines steroidogenic capacities in the ovary, our findings imply that, in immature rats undergoing hormonally induced first ovulation: 1) the early phases of follicular development are supported by androgen production originating from nonfollicular cells; 2) estrogen production in the granulosa cells of Graafian follicles is nourished by a submaximal androgenic output in the theca-interstitial compartments of the ovary.

Oxysterols regulate expression of the steroidogenic acute regulatory protein.

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20alphaOHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20alphaOHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.

Stroke-prone spontaneously hypertensive rats lose their ability to auto-regulate cerebral blood flow prior to stroke.

OBJECTIVES: We hypothesized that the loss of cerebral blood flow (CBF) auto-regulation under hypertensive conditions could promote cerebrovascular over-perfusion and haemorrhage formation. The possibility that CBF auto-regulation becomes defective prior to haemorrhagic stroke development was assessed in Wistar- Kyoto stroke-prone spontaneously hypertensive rats (SHRsp) and related to the myogenic responsiveness of the cerebrovasculature to pressure. METHODS: Laser Doppler techniques were used to measure relative CBF in relation to mean arterial pressure (MAP 130-260 mmHg) within the perfusion domains of the middle (MCA) and posterior (PCA) cerebral arteries. The ability of isolated MCAs and PCAs to constrict to a 120 mmHg pressure step (pressure-dependent constriction) was measured using a pressure myograph. RESULTS: Two weeks prior to stroke, 10-week-old pre-stroke SHRsp exhibited near-constant CBF regulation to a 200 mmHg MAP. Thirteen-week-old pre-stroke SHRsp and age-matched post-stroke SHRsp lost their ability to auto-regulate CBF in the MCA and PCA perfusion domains. CBF increased at a high rate and in a linear manner with MAP. A distinct upper limit to CBF auto-regulation was absent. Pressure-dependent constriction was attenuated prior to stroke, and lost after stroke in isolated MCAs, but not the PCAs, of SHRsp. CONCLUSIONS: The loss of CBF auto-regulation prior to stroke in SHRsp could enhance cerebral perfusion and facilitate the initiation of haemorrhage. Such dysfunction after stroke could produce secondary haemorrhages. Defects in pressure-dependent constriction cannot fully account for the pattern of CBF auto-regulation loss observed in post-stroke SHRsp.

Ethnobotanical-directed discovery of the antihyperglycemic properties of cryptolepine: its isolation from Cryptolepis sanguinolenta, synthesis, and in vitro and in vivo activities.

Using an ethnobotanical approach in combination with in vivo-guided fractionation as a means for lead discovery, cryptolepine was isolated as an antihyperglycemic component of Cryptolepis sanguinolenta. Two syntheses of cryptolepine, including an unambiguous synthesis, are reported. The hydroiodide, hydrochloride, and hydrotrifluoromethanesulfonate (hydrotriflate) salts of cryptolepine were synthesized, and a comparison of their spectral properties and their in vitro activities in a 3T3-L1 glucose transport assay is made. Cryptolepine and its salt forms lower blood glucose in rodent models of type II diabetes. While a number of bioactivities have been reported for cryptolepine, this is the first report that cryptolepine possesses antihyperglycemic properties.

Specific dose-dependent effects of ethane 1,2-dimethanesulfonate in rat and mouse Leydig cells and non-steroidogenic cells on programmed cell death.

The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively kills Leydig cells is poorly understood. To characterize further the cell-specific actions of EDS, we studied biochemical and morphological changes during apoptosis in different Leydig cell and non-steroidogenic cell models. Rat testicular and H540 tumor Leydig cells were killed by 1-2 mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher concentration of EDS was also necessary for activation of apoptosis in non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney cells were resistant. These variable effects of EDS on apoptosis were independent of new protein synthesis and, interestingly, could be delayed by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also observed chromosomal fragmentation and other hallmarks indicative of apoptosis as evidenced by DNA laddering and fluorescent microscopy. Time-lapse photography with a confocal microscope showed that the time of onset, duration and even the sequence of apoptotic events between individual H540 cells was heterogeneous. When the dose of EDS was gradually increased from 2 to 10 mM, the proportion of cells showing normal apoptotic features gradually decreased. Intriguingly, treatment with 10 mM EDS did not result in death for most cells and was marked by an absence of DNA laddering and ultrastructural features of apoptosis and necrosis. However, incubation with 20 mM EDS resulted in necrosis.These results demonstrated that the effects of EDS on cell survival are not specific to Leydig cells, that different cell types have different sensitivities to EDS and that stimulation of the cAMP pathway may mitigate EDS action. The data obtained with H540 cells further revealed that EDS can induce two types of programmed cell death.

Nigericin inhibits accumulation of the steroidogenic acute regulatory protein but not steroidogenesis.

The steroidogenic acute regulatory (StAR) protein mediates the delivery of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol side chain cleavage complex converts it to pregnenolone. While the mechanism by which this mitochondrial protein acts is poorly understood, one component of the mitochondrial electrochemical gradient, the electrochemical potential (DeltaPsi), appears to be essential. In this study, the importance of the other component, the proton gradient (DeltapH), was examined. Disruption of DeltapH with the electroneutral K(+)/H(+) exchanger, nigericin, had no effect on steroidogenesis in MA-10 mouse Leydig tumor cells at concentrations which significantly reduced StAR protein levels. These data indicate for the first time in true steroidogenic cells, that StAR can act prior to being fully imported into the mitochondria and are consistent with observations made in COS-1 cells using mutant forms of StAR. These results support the hypothesis that a DeltaPsi-dependent factor is required for StAR activity and demonstrate that nigericin is the first compound described, capable of inhibiting StAR accumulation without affecting steroidogenesis.

Effects of disruption of the mitochondrial electrochemical gradient on steroidogenesis and the Steroidogenic Acute Regulatory (StAR) protein.

The steroidogenic acute regulatory (StAR) protein, which mediates cholesterol delivery to the inner mitochondrial membrane and the P450scc enzyme, has been shown to require a mitochondrial electrochemical gradient for its activity in vitro. To characterize the role of this gradient in cholesterol transfer, investigations were conducted in whole cells, utilizing the protonophore carbonyl cyanide m-chlorophenylhydrazone (m-CCCP) and the potassium ionophore valinomycin. These reagents, respectively, dissipate the mitochondrial electrochemical gradient and inner mitochondrial membrane potential. Both MA-10 Leydig tumor cell steroidogenesis and mitochondrial import of StAR were inhibited by m-CCCP or valinomycin at concentrations which had only minimal effects on P450scc activity. m-CCCP also inhibited import and processing of both StAR and the truncated StAR mutants, N-19 and C-28, in transfected COS-1 cells. Steroidogenesis induced by StAR and N-47, an active N-terminally truncated StAR mutant, was reduced in transfected COS-1 cells when treated with m-CCCP. This study shows that StAR action requires a membrane potential, which may reflect a functional requirement for import of StAR into the mitochondria, or more likely, an unidentified factor which is sensitive to ionophore treatment. Furthermore, the ability of N-47 to stimulate steroidogenesis in nonsteroidogenic HepG2 liver tumor cells, suggests that the mechanism by which StAR acts may be common to many cell types.

Preclinical development of the Islet Sheet.

The Islet Sheet is a thin planar bioartificial endocrine pancreas fabricated by gelling highly purified alginate and islets of Langerhans. Acellular alginate layers form a uniform immunoprotective barrier to host rejection of the encapsulated cells, with the tissue nourished by passive diffusion from adjacent host tissue. The overall thickness of the Islet Sheet, 250 microm, is chosen to maximize nutrient diffusion. In this paper we describe the early development of the Islet Sheet, including purification and fractionation of the alginates used, difficulties in maintaining sheet planarity, and preliminary metabolic studies in pancreatectomized dogs. In a key experiment, approximately 75,000 allogeneic islet equivalents in six Islet Sheets were sutured to the omentum of a 7-kg female beagle dog at the time of pancreatectomy. Fasting euglycemia was maintained for 84 days. Fed blood sugars were usually below 150 mg/dL. A single injection of 2 U insulin was administered on day 9, and antibiotics were administered for two weeks. No other drugs were used. IVGTT post implant was not normal, but seemed to improve between 30 and 60 days. Upon omentectomy and sheet removal the metabolic parameters deteriorated to a frankly diabetic state within seven days. The sheets did not remain flat, but fragments were recovered within hard, mostly acellular capsules. Dithizone staining showed islets within alginate sheets recovered from the interior of these capsules, suggesting that allogeneic islet tissue survived 84 days and was responsible for maintaining fasting euglycemia.

Beneficial actions of exogenous hyaluronic acid on wound healing.

To determine the effect of exogenous hyaluronic acid (HA) on healing of experimental wounds, responses in the hamster cheek pouch were measured after a hole was cut through the tissue with a biopsy punch. Fluorescence-labeled dextran was administered intravenously as a macromolecular tracer and the microcirculation was observed in vivo with a fluorescence microscope connected to a high-resolution television system. In one group a gelatin sponge soaked in 1.5 ml 16 mg/dl HA in water was applied topically at the time of injury and on postinjury days 1, 3, 5, and 7. The control group received the sponge soaked in the aqueous vehicle. Every 2 days after injury, the microcirculation was observed or histologic specimens were harvested. Wound size decreased almost twice as fast with HA compared with its vehicle (p less than 0.05). Healing was defined as time for total wound closure with at least one microvessel bridging the site of injury and required 16 or more days with vehicle but averaged less than 9 days with HA. Early during healing the repair site was surrounded by widespread extravasation of the fluorescent tracer, an index of inflammation; this area was reduced by two thirds 2 to 4 days after injury with HA compared with its vehicle (p less than 0.05). The density of perfused microvessels was twofold higher with HA 2 to 4 days after injury (p less than 0.05). However, microvessel density was similar in both groups by 6 days after injury and remained similar for at least 45 days after injury, which suggests that HA evoked no unusual angiogenic response. Histologic examination of fixed, stained specimens showed increases in intravascular leukocytes after injury and treatment-related differences in the distribution of intravascular leukocytes in 20 to 40 microns and 40 to 80 microns diameter microvessels 1 to 2 days after injury. Otherwise, leukocyte infiltration during healing was similar in both groups. The mechanism for the beneficial action of HA on healing is unknown. However, several in vitro studies suggest that HA is part of a feedback loop that promotes cell proliferation and migration in actively growing tissues. Alternatively, the role of HA in water homeostasis could favor tissue hydration, which has a well-known beneficial effect on healing.

Masoprocol (nordihydroguaiaretic acid): a new antihyperglycemic agent isolated from the creosote bush (Larrea tridentata).

An ethnomedically-driven approach was used to evaluate the ability of a pure compound isolated from the creosote bush (Larrea tridentata) to lower plasma glucose concentration in two mouse models of type 2 diabetes. The results indicated that plasma glucose concentration fell approximately 8 mmol/l in male C57BL/ks-db/db or C57BL/6J-ob/ob mice following the oral administration of masoprocol (nordihydroguaiaretic acid), a well known lipoxygenase inhibitor. The decline in plasma glucose concentration following masoprocol treatment in the mice was achieved without any change in plasma insulin concentration. In addition, oral glucose tolerance improved and the ability of insulin to lower plasma glucose concentrations was accentuated in masoprocol-treated db/db mice. These data raise the possibility that masoprocol, or other lipoxygenase inhibitors, represents a new approach to the pharmacological treatment of Type 2 diabetes.

A simplified method for approximation of shortages of rural physicians.

The distribution of physicians can be mapped and shortage areas and the number of physicians needed in them can be determined by use of the simple, inexpensive method described. However, the limitations of the methodology must be borne in mind. One should visualize the physician shortage as only a rough indication of the need for primary health care services. More detailed analysis of each area may be required before a new service is actually established, for example, developing a community profile of the planned service area (age sex mix, income, education, race, occupation, and so on), surveying service-level expectation in the community, or studying patients' use of primary care providers in neighboring areas. Even more important may be the selection among a number of possible choices of service alternatives, such as satellite practices, use of physician's assistants or nurse practitioners, or group practices. Estimates based on simplified data and approximations are useful in leading planners to areas of probable undersupply and in helping them to avoid the problems of oversupply. These estimates identify target areas that appear to have physician shortages and point out where more refined analysis should be concentrated.

Biological diversity, indigenous knowledge, drug discovery and intellectual property rights: creating reciprocity and maintaining relationships.

When new plant-derived therapeutics based on indigenous knowledge are being explored, it is important that the pharmaceutical companies return benefits to the native populations and the local governments from which the research material was obtained. When a potentially marketable plant product is being developed, it is essential that equitable agreements have already been established between the pharmaceutical companies and the people and/or countries from which this indigenous knowledge was acquired. Equally important is the commitment to provide immediate reciprocity that will enhance the welfare, the biocultural diversity and the well-being of the forest peoples. These measures should commence when a research project begins and continue during its duration. The development of these measures must be based upon the expressed needs of the indigenous communities. The relationship between the stability of the rain forest biocultural diversity, the creation and development of agro-forest resources and the long term benefits to the forest people is highlighted. Examples of initiatives taken by Shaman Pharmaceuticals Inc. and the Healing Forest Conservancy are described and discussed in the context of exploring appropriate use of intellectual property law to address the ethical issues facing all business and research groups working in the tropics.

Recognizing and responding to adolescent depression.

We have seen that depression is increasingly recognized as a problem which affects adolescents as well as adults. Race by itself is not a factor which increases vulnerability to depression, but the multitude of sociodemographic, biological, and personal factors contributing to the development of depression are not fully understood. Research is needed to better understand how these factors interact in order to better develop prevention strategies and treatment modalities. With regard to treatment, adolescents are truly underserved. There is an awkward fit between the traditional mental health system and the developmental needs of adolescents. Unlike the majority of depressed adults who receive some treatment from general practitioners and medical clinics, adolescents do not tend to visit their pediatricians when they are depressed. One proposed solution is the adolescent comprehensive health care clinic providing a holistic approach to assessment, early intervention, and collaborative treatment between primary provider and mental health worker. Data from our adolescent program suggest that this approach has been successful in dealing with the problem of depression, especially in its early stages. Again, more research is needed to support these preliminary findings, but we believe that the adolescent comprehensive health care approach can be an effective, efficient way of addressing the health needs, mental and physical, of our teens at risk. Federal money for research must be made available, liberalization of health insurance must occur, and state-sponsored services must be developed so that these programs can be made more effective and can be brought to more of the adolescents who need them.